Ribonucleic acids with 4&#39;-thio-modified nucleotides and related methods

ABSTRACT

Disclosed are messenger RNA molecules and related compositions incorporating a 4′-thio modification in the furanose ring of at least one nucleotide residue, and methods of using these mRNAs to produce an encoded therapeutic protein in vivo and to treat or prevent diseases or disorders. In certain embodiments, the 4′-thio modified mRNA provides for enhanced stability and/or reduced immunogenicity in in vivo therapies.

This application claims the benefit under 35 U.S.C. § 119(e) of U.S.patent application No. 61/785,098, filed Mar. 14, 2013, the entirety ofwhich is incorporated herein by reference.

BACKGROUND

The present invention relates to messenger ribonucleic acids (mRNAs)comprising 4′-thio-modified nucleotide residues, compositions comprisingthose mRNAs, and methods of making and using same.

Gene therapy using messenger RNA has been proposed as an approach forthe treatment of a variety of diseases. The concept of introduction ofmessenger RNA (mRNA) as a means of protein production within a host hasbeen reported previously. Yamamoto, A. et al. Eur. J. Pharm. 71:484-489(2009); Debus, H. et al. J. Control Rel. 148:334-343 (2010). However,successful administration of mRNA for in vivo protein productiontypically required mRNA being packaged (such as, e.g., mRNA complexedwith a polymer or lipid carrier). See, e.g., International Patent Appl.Publ. Nos. WO 2011/06810 and WO 2012/170930. Administration ofunpackaged (naked) mRNA required chemically-modified nucleotides to beincorporated within an mRNA to result in a more stable and beneficialtherapeutic. See, e.g., M. Kormann et al. Nature Biotech. 29:154-159(2011); K. Kariko, Molecular Therapy 16(11):1833-1840 (2008).

The administration of mRNAs encoding a therapeutic protein that can beproduced in vivo may provide significant advantages over administrationof DNA encoding the therapeutic protein as well as direct administrationof the therapeutic protein. However, while the development oftherapeutic mRNAs encoding therapeutic proteins represents a promisingadvancement in medical therapies, the utility of such treatments canstill be limited by the poor stability of mRNAs in vivo, particularlythose encoding full length proteins.

In particular, poor stability of mRNAs used in gene replacement therapycan result in insufficient or less optimal production of the encodedtherapeutic protein in vivo. Following the administration of an mRNAthat encodes a therapeutic protein, the mRNA may undergo degradation,for example upon exposure to one or more nucleases in vivo.Ribonucleases (e.g., endoribonucleases and exoribonucleases) represent aclass of nuclease enzymes that are capable of catalyzing the degradationof RNA into smaller components and thereby render the mRNA unable toproduce the therapeutic protein. Nuclease enzymes (e.g., RNase) aretherefore capable of shortening the circulatory half-life of, forexample, synthetically or recombinantly-prepared mRNAs. Followingnucleolytic degradation, an mRNA is not translated, and thus, isprevented from exerting an intended therapeutic benefit, which cansignificantly reduce the efficacy of the mRNA gene therapy.

SUMMARY

The present invention provides an improved modified mRNA for morestable, robust and sustained in vivo protein production. The presentinvention is based, in part, on the realization that the stability ofmRNA used to produce therapeutic proteins in vivo can be furtherimproved by incorporating modified ribonucleotides in which the 4′oxygen in the ribose moiety is substituted by a sulfur. Althoughsubstitution of the 4′ oxygen in the ribose moiety of ribonucleotideswith a sulfur has been reported previously by S. Hoshika et al. (Nuc.Ac. Res. Supp. 3:209-210 (2003)) and M. Takahashi, M. et al. (Nuc. Ac.Res. 37:1353-1362 (2009)), both reports involved short syntheticsegments of RNA containing 4′-thio residues of at most 15 residues inlength for RNA interference; short RNAs of 19-21 residues comprising4′-thio-modified nucleotides have also been reported for RNAinterference (Dande et al., J. Med. Chem. 49:1624-1634 (2006)) and fordeveloping aptamers (up to 59 residues in length; Hoshika et al., Nuc.Ac. Res. 32:3815-3825(2004); Kato et al., Nuc. Ac. Res. 33:2942-2951(2005); Minakawa et al., Bioorg. Med. Chem. 16:9450-9456 (2008)). Thesereports, however, are not predictive of the effect of incorporating4′-thioribonucleotides into a full length mRNA (that is, an mRNAencoding a full length functional therapeutic protein and optionallycontaining one or more noncoding regions), which generally has a lengthmuch longer than any of the interfering RNAs or aptamers tested in theprior art and does not exist in a uniformly duplexed state and may adopta conformation with a large non-helical and/or single-strandedcomponent. More importantly, it was unclear if mRNAs incorporating4′-thio-modified nucleotides could be successfully used for in vivoprotein production prior to the present invention. As described hereinincluding the examples, the present inventors have successfullysynthesized full length mRNAs incorporating one or more 4′-thio-modifiednucleotides (e.g., 4′-thio-ATPs, 4′-thio-UTPs, 4′-thio-GTPs, and/or4′-thio-CTPs). Despite the concern over the length of mRNAs, the presentinventors were able to synthesize full length mRNAs incorporating up to100% 4′-thio-ATPs, 100% 4′-thio-UTPs, 100% 4′-thio-GTPs, and/or 100%4′-thio-CTPs. As shown in the Examples, such modified mRNAs are morestable than unmodified mRNAs and surprisingly, such extensivemodifications do not appear to impact the ability of modified mRNAs tobe effectively translated in vivo.

Accordingly, the present invention provides mRNAs that allow bettercontrol over, for example, the stability, immunogenicity, andtranslational efficiency of the mRNA, and compositions comprising thosemRNAs and, optionally, a carrier, as well as methods of using thosemRNAs and compositions to induce expression of a therapeutic protein invivo for treatment of diseases and/or disorders.

In some embodiments, the invention provides an mRNA molecule having acoding region and optionally, one or more non-coding regions, whereinthe mRNA comprises at least one nucleotide residue that incorporates a4′-thio-substituted furanose ring. In some embodiments, a provided mRNAcontains at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%nucleotide residues that incorporate a 4′-thio-substituted furanosering. In some embodiments, a provided mRNA contains 100% nucleotideresidues that incorporate a 4′-thio-substituted furanose ring. In someembodiments, a provided mRNA contains up to 5%, 10%, 15%, 20%, 25%, 30%,35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%,97%, 98%, 99% or 100% 4′-thio-ATPs. In some embodiments, a provided mRNAcontains up to 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%,60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%4′-thio-UTPs. In some embodiments, a provided mRNA contains up to 5%,10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%,80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% 4′-thio-GTPs. In someembodiments, a provided mRNA contains up to 5%, 10%, 15%, 20%, 25%, 30%,35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%,97%, 98%, 99% or 100% 4′-thio-CTPs. In some embodiments, a provided mRNAcontains a combination of various 4′-thio-modified NTPs describedherein.

In some embodiments, a provided mRNA comprises a non-coding region. Insome embodiments, a provided mRNA comprises a poly-A and/or a poly-Utail. In some embodiments, a provided mRNA comprises a 5′ cap structure.

In some embodiments, a provided mRNA further comprises at least onenonstandard nucleotide residue. In some embodiments, the at least onenonstandard nucleotide residue is chosen from one or more of5-methyl-cytidine, pseudouridine, and 2-thio-uridine. In someembodiments, the at least one nonstandard nucleotide residueincorporates a 4′-thio-furanose ring. In some embodiments, up to 5%,10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%,80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% nonstandard nucleotideresidues incorporate a 4′-thio-furanose ring.

In some embodiments, a provided mRNA is at least 60 residues in length.In some embodiments, a provided mRNA is at least about 70, about 80,about 90, about 100, about 150, about 200, about 300, about 400, about500, about 1,000, about 1,500, about 2,000, about 2,500, about 3,000,about 3,500, about 4,000, about 4,500, or about 5,000 residues inlength.

Additional embodiments of the invention provide compositions comprisingat least one mRNA molecule having a coding region and optionally, one ormore non-coding regions, wherein the mRNA comprises at least onenucleotide residue that incorporates a 4′-thio-substituted furanose ringand a carrier. In some embodiments, a provided composition comprises atleast one mRNA having a coding region and optionally, one or morenon-coding regions, wherein the mRNA comprises at least one nucleotideresidue that incorporates a 4′-thio-substituted furanose ring and acarrier, and the mRNA is at least 60 residues in length. In certainembodiments, the compositions of the invention comprise at least onemRNA molecule having a coding region and optionally, one or morenon-coding regions, wherein the mRNA comprises at least one nucleotideresidue that incorporates a 4′-thio-substituted furanose ring, and iscomplexed with a polymer based carrier or a lipid nanoparticle.

The invention further provides methods of producing a therapeuticprotein in vivo, comprising administering to a subject at least one mRNAmolecule having a coding region and optionally, one or more non-codingregions, wherein the mRNA comprises at least one nucleotide residue thatincorporates a 4′-thio-substituted furanose ring, or a compositioncomprising such mRNA and a carrier. The invention also provides methodsof treating a subject in need of a therapeutic protein, comprisingadministering at least one mRNA molecule having a coding region andoptionally, a non-coding region, wherein the mRNA comprises at least onenucleotide residue that incorporates a 4′-thio-substituted furanosering, or a composition comprising such mRNA and a carrier. In someembodiments, an administered mRNA in a provided method is at least 60residues in length. Various modified mRNAs described herein may be usedfor production of therapeutic proteins or for treatment of variousdiseases, disorders or conditions.

In some embodiments, the present invention provides a method forproducing a protein using a modified mRNA described herein. Such amethod of protein production may be used in an in vitro cell freesystem, in vitro cell based system, or in vivo system. In variousembodiments, a suitable mRNA comprises at least one nucleotide residuethat incorporates a 4′-thio-substituted furanose ring. In someembodiments, a suitable mRNA comprises up to about 5%, 10%, 15%, 20%,25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,95%, 96%, 97%, 98%, 99%, or 100% of nucleotide residues (e.g., ATP, CTP,GTP, UTP, and/or non-standard NTPs) that incorporate a4′-thio-substituted furanose ring. In some embodiments, a provided mRNAcomprises a poly(A) or poly(U) tail. In some embodiments, a providedmRNA is at least 60 residues in length.

In some embodiments, the present invention provides use of a providedmRNA molecule for the manufacture of a medicament that is capable ofproducing a therapeutic protein in vivo.

In some other embodiments, the present invention provides a method formaking a provided mRNA. In some other embodiments, the present inventionprovides a method for in vitro synthesis of a provided mRNA. In someother embodiments, the present invention provides a method for making(e.g., in vitro synthesizing) a provided mRNA containing up to about 5%,10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%,80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of nucleotide residues(e.g., ATP, CTP, GTP, UTP, and/or non-standard NTPs) that incorporate a4′-thio-substituted furanose ring. In some embodiments, the presentinvention provides a method for making (e.g., in vitro synthesizing) aprovided mRNA at least about 60, about 70, about 80, about 90, about100, about 150, about 200, about 300, about 400, about 500, about 1,000,about 1,500, about 2,000, about 2,500, about 3,000, about 3,500, about4,000, about 4,500, or about 5,000 residues in length.

Additional objects and advantages of the invention will be set forth inpart in the description which follows, and in part will be obvious fromthe description, or may be learned by practice of the invention. Theobjects and advantages of the invention will be realized and attained bymeans of the elements and combinations particularly pointed out in theappended claims.

It is to be understood that both the foregoing general description andthe following detailed description are exemplary and explanatory onlyand are not restrictive of the invention, as claimed. The accompanyingdrawing, which is incorporated in and constitutes a part of thisspecification, illustrates several embodiments of the invention andtogether with the description, serves to further explain the principlesof the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

The drawings are for illustration purposes only, not for limitation.

FIG. 1 shows molecular structures of exemplary 4′-thioRNA bases:4′-thio-adenosine, 4′-thio-guanosine, 4′-thio-cytidine, 4′-thio-uridine,4′-thio-5-methyl-cytidine, 4′-thio-pseudouridine, and4′-thio-2-thiouridine.

FIG. 2 shows luciferase detection from FFL luciferase production in HEK293T cells post-transfection of modified and unmodified FFL mRNA.

FIG. 3 shows the results of a stability study of modified and unmodifiedFFL mRNA.

FIG. 4 shows luciferase detection from FFL luciferase production inmouse liver post-administration of modified and unmodified FFL mRNA.

DETAILED DESCRIPTION OF THE INVENTION

As used herein, the term “mRNA” is used to refer to modified and/orunmodified RNA including a coding region and, optionally, a noncodingregion. The term “coding region” refers to a portion or region of themRNA that can be translated into a chain of amino acids, i.e., two ormore amino acids linked by peptide bonds. A chain of amino acids is alsoreferred to as a peptide or a polypeptide, which can fold into a protein(e.g., a therapeutic protein). The term “noncoding region” refers to aportion or region of the mRNA that are typically not translated.Noncoding region typically includes 5′ untranslated region and/or 3′untranslated region including but not limited to a poly(A) or poly(U)tail.

A “nonstandard nucleobase” is a base moiety other than the natural basesadenine (A), cytosine (C), guanine (G), thymine (T), or uracil (U). Thenonstandard nucleobase is an analog of a specific nucleobase (A, C, G,T, or U) when its base pairing properties in a nucleic acid double helixand locus of incorporation by DNA or RNA polymerases in a nucleic aciddouble helix (including a local RNA-DNA helix such as that formed duringtranscription of a DNA template by RNA polymerase) are most similar toone of the five previously listed nucleobases, with the exception thatanalogs of T will generally also be analogs of U and vice versa. Theterm “nonstandard” used in conjunction with terms including but notlimited to “nucleoside,” “base,” “nucleotide,” or “residue” is to beinterpreted in the same manner as if it were used in conjunction with“nucleobase.”

As used herein, the term “therapeutic protein” includes any proteinthat, if administered to a subject, provides a beneficial effect on thehealth and well-being of the subject. In some embodiments, a deficiency,lack of, or aberrant expression of that protein in a subject gives riseto a disease or condition. “Therapeutic protein” may also refer to aprotein that is not normally present or is not normally present insufficient quantities in a subject to achieve a desired therapeuticeffect.

The term “helper lipid” as used herein refers to any neutral orzwitterionic lipid material including cholesterol. Without wishing to beheld to a particular theory, helper lipids may add stability, rigidity,and/or fluidity within lipid bilayers/nanoparticles.

The mRNAs of the invention employ specific chemically-modified bases, inwhich the 4′ oxygen in the ribose moiety of a nucleotide residue isreplaced with sulfur, for substitution into a messenger ribonucleic acidmolecule to enhance its biological properties upon administration to asubject. Exemplary 4′-thio modified nucleotide residues forincorporation into an mRNA of the invention are depicted in FIG. 1(showing modified nucleotide residues containing a thio-substitutedfuranose ring). In some embodiments, 4′-thio modification of thefuranose ring provides improved resistance to exonucleases,endonucleases, and/or other RNA degradation enzymes in human serum. Suchstability can afford an increased RNA half-life. Thus, for example,administration of an mRNA having a 4′-thio modification in the furanosering or a composition comprising such mRNA results in cellular uptake ofan mRNA having improved biological properties, e.g., increasedhalf-life, which in turn contributes to increased protein production invivo.

In certain embodiments, at least 1% of the adenosine nucleotide residuesin the RNA have a 4′-thio modification in the furanose ring. Forexample, about 1-5%, 5-15%, 15-30%, 30-50%, 50-75%, 75-90%, 90-99%, or99-100% of the adenosine in the mRNA can be 4′-thio-adenosine.

In some embodiments, at least about 1%, about 5%, about 10%, about 15%,about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%,about 85%, about 90%, about 95%, about 96%, about 97%, about 98% orabout 99% of the adenosine residues in the mRNA are 4′-thio-adenosine.In some embodiments, about 100% of the adenosine residues in the mRNAare 4′-thio-adenosine.

In certain embodiments, at least 1% of the guanosine nucleotide residuesin the RNA have a 4′-thio modification in the furanose ring. Forexample, about 1-5%, 5-15%, 15-30%, 30-50%, 50-75%, 75-90%, 90-99%, or99-100% of the guanosine in the mRNA can be 4′-thio-guanosine.

In some embodiments, at least about 1%, about 5%, about 10%, about 15%,about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%,about 85%, about 90%, about 95%, about 96%, about 97%, about 98% orabout 99% of the guanosine residues in the mRNA are 4′-thio-guanosine.In some embodiments, about 100% of the guanosine residues in the mRNAare 4′-thio-guanosine.

In certain embodiments, at least 1% of the uridine nucleotide residuesin the RNA have a 4′-thio modification in the furanose ring. Forexample, about 1-5%, 5-15%, 15-30%, 30-50%, 50-75%, 75-90%, 90-99%, or99-100% of the uridine in the mRNA can be 4′-thio-uridine.

In some embodiments, at least about 1%, about 5%, about 10%, about 15%,about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%,about 85%, about 90%, about 95%, about 96%, about 97%, about 98% orabout 99% of the uridine residues in the mRNA are 4′-thio-uridine. Insome embodiments, about 100% of the uridine residues in the mRNA are4′-thio-uridine.

In certain embodiments, at least 1% of the cytidine nucleotide residuesin the RNA have a 4′-thio modification in the furanose ring. Forexample, about 1-5%, 5-15%, 15-30%, 30-50%, 50-75%, 75-90%, 90-99%, or99-100% of the cytidine in the mRNA can be 4′-thio-cytidine.

In some embodiments, at least about 1%, about 5%, about 10%, about 15%,about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%,about 85%, about 90%, about 95%, about 96%, about 97%, about 98% orabout 99% of the cytidine residues in the mRNA are 4′-thio-cytidine. Insome embodiments, about 100% of the cytidine residues in the mRNA are4′-thio-cytidine.

In some embodiments, each 4′-thio-modified nucleotide in a provided mRNAis 4′-thio-uridine. In some embodiments, each 4′-thio-modifiednucleotide in a provided mRNA is 4′-thio-cytidine. In some embodiments,each 4′-thio-modified nucleotide in a provided mRNA is independently4′-thio-uridine or 4′-thio-cytidine. In some embodiments, a providedmRNA comprises at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60,65, 70, 75, 80, 85, 90, 95, 100 or more 4′-thio-uridine or4′-thio-cytidine. In some embodiments, a provided mRNA comprises atleast one 4′-thio-adenosine residue. In some embodiments, a providedmRNA comprises at least one 4′-thio-guanosine residue. In someembodiments, a provided mRNA comprises at least one 4′-thio-guanosine or4′-thio-adenosine residue. In some embodiments, a provided mRNAcomprises at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65,70, 75, 80, 85, 90, 95, 100 or more 4′-thio-guanosine or4′-thio-adenosine residues.

In certain embodiments, the fraction of nucleotide residues with a4′-thio modification in the furanose ring of one base type (e.g.,adenosine, guanosine, uridine, or cytidine) varies independently of thefraction of modified nucleotide residues of the other base types.

In certain embodiments, less than 10% of the nucleotide residues have a4′-thio modification in the furanose ring. For example, about 1-5%,5-10%, 3-5%, 1-3%, 0.1-1%, or 0.01-0.1% of the nucleotide residues canincorporate a 4′-thio-substituted furanose ring.

In other embodiments, more than 10% of the nucleotide residues have a4′-thio modification in the furanose ring. For example, about 10-15%,15-20%, 20-25%, 25-30%, 30-35%, 35-40%, 40-45% or 45-50% of thenucleotide residues can incorporate a 4′-thio-substituted furanose ring.In some embodiments, more than 50% of the nucleotide residues have a4′-thioRNA modification in the furanose ring. For example, about 50-55%,55-60%, 60-65%, 65-70%, 70-75%, 75-80%, 80-85%, 85-90%, 90-95%, 95-100%,95-97%, 97-98%, 98-99%, 99-99.9%, or 99.9-100% of the nucleotideresidues incorporate a 4′-thio-substituted furanose ring.

In some embodiments, at least about 1%, 5%, 10%, 15%, 20%, 25%, 30%,35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%,97%, 98%, or 99% of the nucleotide residues can incorporate a4′-thio-substituted furanose ring. In some embodiments, about 100%nucleotide residues can incorporate a 4′-thio-substituted furanose ring.

The coding and non-coding regions in the mRNAs of the invention mayencompass non-contiguous regions of sequence. The optional non-codingregions may include one or more of a 5′ untranslated region (UTR), a 3′UTR, a poly-A, poly-U or poly-C tail, and/or a 5′ cap structure. In someembodiments, a provided mRNA comprises a non-coding region. In someembodiments, a provided mRNA comprises a 5′ UTR. In some embodiments, aprovided mRNA comprises a 3′ UTR. In some embodiments, a provided mRNAcomprises a 5′ cap structure. In some embodiments, a provided mRNAcomprises a poly-A tail. In some embodiments, a provided mRNA comprisesa 5′-UTR sequence, a 3′-UTR sequence and a poly-A tail. In someembodiments, a provided mRNA comprises a 5′-UTR sequence, a codingregion, a 3′-UTR sequence and a poly-A tail. In some embodiments, aprovided mRNA comprises a 5′-UTR sequence, a 5′ cap, a 3′-UTR sequenceand a poly-A tail. In some embodiments, a provided mRNA comprises a5′-UTR sequence, a 5′ cap, a coding region, a 3′-UTR sequence and apoly-A tail.

In certain embodiments, the poly-A, poly-U or poly-C tail comprisesnucleotide residues that incorporate a 4′-thio-substituted furanosering. In some embodiments, only the poly-A, poly-U or poly-C tail orother components of the non-coding region incorporate nucleotideresidues having a 4′-thio substitution in the furanose ring, while theremainder of the nucleotide residues in the mRNA molecule do not containa 4′-thio-furanose modification. In some embodiments, the coding regioncomprises nucleotide residues that incorporate a 4′-thio-substitutedfuranose ring. In certain embodiments, both the coding and non-codingregions (if present) incorporate nucleotide residues having a 4′-thiosubstitution in the furanose ring. In certain embodiments, the length ofthe poly-A, poly-U or poly-C tail may vary. For example, the length ofthe poly-A, poly-U, or poly-C tail may be at least about 50, 70, 90,100, 150, 200, 250, 300, 400, or 500 nucleotides in length. In someembodiments, the length of the poly-A, poly-U or poly-C tail is lessthan about 90, 100, 150, 200, 250, 300, 400, or 500 nucleotides inlength. In certain embodiments, the mRNA molecule may includemodifications in addition to a 4′-thio-substituted furanose ring. Forexample, the molecule may incorporate any nonstandard nucleobase.Certain embodiments may include nucleotide residue modifications such as5-methyl-cytidine (“5mC”), pseudouridine (“ψU”), 2-thio-uridine (“2sU”),5-methylcytosine, isocytosine, pseudoisocytosine, 5-bromouracil,5-propynyluracil, 6-aminopurine, 2-aminopurine, inosine, diaminopurineand 2-chloro-6-aminopurine cytosine as well as combinations of thesemodifications and other nucleotide residue modifications. Certainembodiments may further include additional modifications to the furanosering or other parts of the nucleotide residue, e.g., the nucleobase. Forexample, in some embodiments, a 4′-thio substituted furanose ring can beincluded within an unmodified or a modified base such as, e.g.,pseudouridine, 2-thiouridine, and 5-methylcytidine. In certainembodiments, any of these modifications may be present in 0-100% of thenucleotide residues—for example, more than 0%, 1%, 10%, 50%, 90% or 95%,or 100% of the nucleotide residues individually or in combination. Insome embodiments, a provided mRNA comprises at least one nonstandardnucleotide residue. In some embodiments, the at least one nonstandardnucleotide residue is chosen from one or more of 5-methyl-cytidine,pseudouridine, and 2-thio-uridine. In some embodiments, the at least onenonstandard nucleotide residue in 5-methyl-cytidine. In someembodiments, the at least one nonstandard nucleotide residueincorporates a 4′-thio-furanose ring. In some embodiments, up to 5%,10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%,80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% nonstandard nucleotideresidues incorporate a 4′-thio-furanose ring.

Additional modifications may include, for example, sugar modificationsor substitutions (e.g., one or more of a 2′-O-alkyl modification, alocked nucleic acid (LNA)). In embodiments where the sugar modificationis a 2′-O-alkyl modification, such modification may include, but are notlimited to a 2′-deoxy-2′-fluoro modification, a 2′-O-methylmodification, a 2′-O-methoxyethyl modification and a 2′-deoxymodification.

In certain embodiments, 0-100% of the mRNA may be single-stranded. Incertain embodiments, 0-100% of the RNA may adopt a non-helicalconformation.

In certain embodiments, the compositions of the invention comprise mRNAsin which about 100% of the uridine residues are replaced with4′-thio-uridine.

In certain embodiments, the compositions of the invention comprise mRNAsin which about 100% of the uridine residues are replaced with4′-thio-uridine and about 100% of the cytidine residues are replacedwith 5-methyl-cytidine.

In certain embodiments, the compositions of the invention comprise mRNAsin which about 100% of the uridine residues are replaced with4′-thio-pseudouridine.

In certain embodiments, the compositions of the invention comprise mRNAsin which about 100% of the uridine residues are replaced with4′-thio-pseudouridine and about 100% of the cytidine residues arereplaced with 5-methyl-cytidine.

In some embodiments, a provided mRNA provides a beneficial biologicaleffect, for example but not limited to increased stability, improvedprotein production rate, and/or higher protein yield, when compared witha corresponding natural mRNA. In some embodiments, a provided mRNA hasincreased stability (e.g., a longer serum half-life) when administeredin vivo, as compared with a corresponding natural mRNA (i.e., acorresponding mRNA without modification).

The mRNA of the invention can be more resistant to nuclease (e.g.,endonuclease) degradation to an extent that results in an increase inthe amount of the therapeutic protein translated from the mRNAtranscript upon administration to a subject by at least about 2.5%, 5%,7.5%, 10%, 15%, 20%, 25%, 30%, 33%, 36%, 40%, 50%, 60%, 70%, 75%, 80%,90%, 95%, 99%, 100%, 110%, 120%, 125%, 150%, 175%, 200%, 250%, 300%,400%, 500%, 600%, 700%, 750%, 800%, 900%, or 1,000%, as compared to acorresponding mRNA without modification.

In certain embodiments, the length of the modified mRNA molecule in thecompositions of the invention is at least 200 nucleotide residues inlength. For example, the mRNA may be at least about 200, 300, 400, 500,1000, 2000, 3000, 4000, or 5000 nucleotide residues in length. In someembodiments, a provided mRNA is at least 60 residues in length. In someembodiments, a provided mRNA is at least about 70, about 80, about 90,about 100, about 150, about 200, about 300, about 400, about 500, about600, about 700, about 800, about 900, about 1,000, about 1,500, about2,000, about 2,500, about 3,000, about 4,000, about 5,000, about 6,000or about 7000 residues in length.

In some embodiments of the invention, the therapeutic protein encoded bythe mRNAs of the invention may be any protein where a deficiency, lackof, or aberrant expression of that protein gives rise to a diseaseand/or condition. In some embodiments, the therapeutic protein may be anenzyme. In other embodiments, the therapeutic protein is one that is notnormally present or is not normally present in sufficient quantities ina subject to achieve the desired therapeutic effect.

For example, a non-limiting selection of suitable therapeutic proteinsincludes erythropoietin, insulin, human growth hormone, cystic fibrosistransmembrane conductance regulator (CFTR), insulin, alpha-galactosidaseA, alpha-L-iduronidase, iduronate-2-sulfatase,N-acetylglucosamine-1-phosphate transferase, N-acetylglucosaminidase,alpha-glucosaminide acetyltransferase, N-acetylglucosamine 6-sulfatase,N-acetylgalactosamine-4-sulfatase, beta-glucosidase, galactose-6-sulfatesulfatase, beta-galactosidase, beta-glucuronidase, glucocerebrosidase,heparan sulfamidase, hyaluronidase, galactocerebrosidase, ornithinetranscarbamylase (OTC), carbamoyl-phosphate synthetase 1 (CPS1),argininosuccinate synthetase (ASS1), argininosuccinate lyase (ASL),arginase 1 (ARG1), glucose-6-phosphatase, glucose-6-phosphatetranslocase, glycogen debranching enzyme, lysosomal alpha-glucosidase,1,4-alpha-glucan branching enzyme, glycogen phosphorylase,phosphofructokinase, liver phosphorylase, GLUT-2, UDP glycogen synthase,alpha-L-iduronidase, iduronate sulfate silfatase, heparan sulfatesulfamidase, alpha-N-acetylglucose amidase,alpha-glucosaminid-N-acetyltransferase, N-acetylglucosamine-6-sulfatesulfatase, apolipoprotein E, low density lipoprotein receptor (LDLR),clotting factors, such as, e.g., Factor VIII, and Factor IX, spinalmotor neuron 1 (SMN1), phenylalanine hydroxylase, propionyl-CoAcarboxylase, porphobilinogen deaminase, methylmalonyl-CoA mutase, urateoxidase, C1 esterase inhibitor, and acid alpha-glucosidase.

In certain embodiments, the mRNA molecules of the invention may beadministered as naked or unpackaged mRNA. In some embodiments, theadministration of the mRNA in the compositions of the invention may befacilitated by inclusion of a suitable carrier. In certain embodiments,the carrier is selected based upon its ability to facilitate thetransfection of a target cell with one or more mRNAs.

As used herein, the term “carrier” includes any of the standardpharmaceutical carriers, vehicles, diluents, excipients and the likewhich are generally intended for use in connection with theadministration of biologically active agents, including mRNA. Thecompositions and, in particular, the carriers described herein arecapable of delivering and/or stabilizing mRNA of varying sizes to theirtarget cells or tissues. In certain embodiments, the compositions of theinvention comprise carriers that are capable of delivering large mRNAs(e.g., mRNAs of at least 5 kDa, 10 kDa, 12 kDa, 15 kDa, 20 kDa, 25 kDa,30 kDa, or more, or of at least 60, 70, 80, 90, 100, 150, 200, 300, 400,500, 600, 700, 800, 900, 1,000, 1,500, 2,000, 2,500, 3,000, 3,500,4,000, 4,500, 5,000, 5,500, 6,000, 6,500, or 7,000 residues in length).mRNAs according to the present invention may be synthesized according toany of a variety of known methods. For example, mRNAs according to thepresent invention may be synthesized via in vitro transcription (IVT).Briefly, IVT is typically performed with a linear or circular DNAtemplate containing a promoter, a pool of ribonucleotide triphosphatesincluding desired amount(s) of 4′-thio-modified standard and/ornon-standard ribonucleotides (e.g., one or more desired 4′-thio-NTP(s))and optionally, mixed with unmodified ribonucleotide triphosphates, abuffer system that may include DTT and magnesium ions, and anappropriate RNA polymerase (e.g., T3, T7 or SP6 RNA polymerase), DNAseI, pyrophosphatase, and/or RNAse inhibitor. The exact conditions willvary according to the specific application. It is observed that4′-thio-modified standard and/or non-standard ribonucleotides may beeffectively incorporated into a full length mRNA of any length.

In some embodiments, for the preparation of mRNA according to theinvention, a DNA template is transcribed in vitro. A suitable DNAtemplate typically has a promoter, for example a T3, T7 or SP6 promoter,for in vitro transcription, followed by desired nucleotide sequence forencoding a protein of interest and a termination signal. Typically, mRNAsynthesis includes the addition of a “cap” on the N-terminal (5′) end,and a “tail” on the C-terminal (3′) end. The presence of the cap isimportant in providing resistance to nucleases found in most eukaryoticcells. The presence of a “tail” serves to protect the mRNA fromexonuclease degradation.

Thus, in some embodiments, mRNAs according to the present inventioninclude a 5′ cap structure. A 5′ cap is typically added as follows:first, an RNA terminal phosphatase removes one of the terminal phosphategroups from the 5′ nucleotide, leaving two terminal phosphates;guanosine triphosphate (GTP) is then added to the terminal phosphatesvia a guanylyl transferase, producing a 5′5′5 triphosphate linkage; andthe 7-nitrogen of guanine is then methylated by a methyltransferase.Examples of cap structures include, but are not limited to, m7G(5′)ppp(5′(A,G(5′)ppp(5′)A and G(5′)ppp(5′)G.

In some embodiments, mRNAs according to the present invention include a3′ tail structure. A suitable 3′ tail structure includes, but is notlimited to, a poly-A, poly-U and/or poly-C tail. Exemplary suitablepoly-A, poly-U and poly-C tails are described above. The poly-U orpoly-C tail may be added to the poly-A tail or may substitute the poly-Atail.

In some embodiments, mRNAs according to the present invention include a5′ and/or 3′ untranslated region. In some embodiments, a 5′ untranslatedregion includes one or more elements that affect an mRNA's stability ortranslation, for example, an iron responsive element. In someembodiments, a 5′ untranslated region may be between about 50 and 500nucleotides in length (e.g., about 50 and 400 nucleotides in length,about 50 and 300 nucleotides in length, about 50 and 200 nucleotides inlength, or about 50 and 100 nucleotides in length).

In certain embodiments of the present invention, the carrier may beselected and/or prepared to optimize delivery of the mRNA to a targetcell, tissue or organ. For example, if the target cell is a pneumocytethe properties of the carrier (e.g., size, charge and/or pH) may beoptimized to effectively deliver such carrier to the target cell ororgan, reduce immune clearance, and/or promote retention in that targetorgan. Alternatively, if the target tissue is the central nervous system(e.g., to facilitate delivery of mRNA polynucleotides to targeted brainregion(s) or spinal tissue) selection and preparation of the carriermust consider penetration of, and retention within, the blood brainbarrier and/or the use of alternate means of directly delivering suchcarrier to such target tissue. In certain embodiments, the compositionsof the present invention may be combined with agents that facilitate thetransfer of exogenous polynucleotides from the local tissues or organsinto which such compositions were administered to one or more peripheraltarget organs or tissues.

In certain embodiments, the carriers employed in the compositions of theinvention may comprise a liposomal vesicle, or other means to facilitatethe transfer of an mRNA to target cells and tissues. Suitable carriersinclude, but are not limited to, polymer based carriers, such aspolyethyleneimine (PEI), lipid nanoparticles and liposomes,nanoliposomes, ceramide-containing nanoliposomes, proteoliposomes, bothnatural and synthetically-derived exosomes, natural, synthetic andsemi-synthetic lamellar bodies, nanoparticulates, calciumphosphor-silicate nanoparticulates, sol-gels, calcium phosphatenanoparticulates, silicon dioxide nanoparticulates, nanocrystallineparticulates, semiconductor nanoparticulates, poly(D-arginine),nanodendrimers, starch-based delivery systems, micelles, emulsions,niosomes, plasmids, viruses, calcium phosphate nucleotides, aptamers,peptides and other vectorial tags. Also contemplated is the use ofbionanocapsules and other viral capsid proteins assemblies as suitablecarriers. (Hum. Gene Ther. 19(9):887-95 (2008)).

In certain embodiments of the invention, the carrier is formulated usinga polymer as a carrier, alone or in combination with other carriers.Suitable polymers may include, for example, polyacrylates,polyalkycyanoacrylates, polylactide, polylactide-polyglycolidecopolymers, polycaprolactones, dextran, albumin, gelatin, alginate,collagen, chitosan, cyclodextrins, protamine, PEGylated protamine, PLL,PEGylated PLL and polyethylenimine (PEI), including, but not limited tobranched PEI (25 kDa). In some embodiments, a polymer may be one or moremulti-domain-block polymers. In some embodiments, a polymer may comprisea dry powder formulation of the polymer or polymers.

The use of liposomal carriers to facilitate the delivery ofpolynucleotides to target cells is also contemplated by the presentinvention. Liposomes (e.g., liposomal lipid nanoparticles) are generallyuseful in a variety of applications in research, industry, and medicine,particularly for their use as carriers of diagnostic or therapeuticcompounds in vivo (Lasic et al., Trends Biotechnol., 16:307-321 (1998);Drummond et al., Pharmacol. Rev., 51:691-743 (1999)) and are usuallycharacterized as microscopic vesicles having an interior aqua spacesequestered from an outer medium by a membrane of one or more bilayers.Bilayer membranes of liposomes are typically formed by amphiphilicmolecules, such as lipids of synthetic or natural origin that comprisespatially separated hydrophilic and hydrophobic domains. Bilayermembranes of the liposomes can also be formed by amphiphilic polymersand surfactants (e.g., polymerosomes, niosomes, etc.).

In certain embodiments, the mRNA molecules is complexed with lipidnanoparticles to facilitate delivery to the target cell. Examples ofsuitable lipids include, for example, the phosphatidyl compounds (e.g.,phosphatidylglycerol, phosphatidylcholine, phosphatidylserine,phosphatidylethanolamine, sphingolipids, cerebrosides, andgangliosides). In certain embodiments, the mRNA molecules andcompositions of the invention may be combined with a multi-componentlipid mixture of varying ratios employing one or more cationic lipids,helper lipids and PEGylated lipids designed to encapsulate variousnucleic acid-based materials.

Cationic lipids may include, but are not limited to DOTAP(1,2-dioleyl-3-trimethylammonium propane), DODAP(1,2-dioleyl-3-dimethylammonium propane), cKK-E12(3,6-bis(4-(bis(2-hydroxydodecyl)amino)butyl)piperazine-2,5-dione),dialkylamino-based, imidazole-based, guanidinium-based, XTC(2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane), MC3(((6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl4-(dimethylamino)butanoate), ALNY-100((3aR,5s,6aS)-N,N-dimethyl-2,2-di((9Z,12Z)-octadeca-9,12-dienyl)tetrahydro-3aH-cyclopenta[d][1,3]dioxol-5-amine)),NC98-5(4,7,13-tris(3-oxo-3-(undecylamino)propyl)-N1,N16-diundecyl-4,7,10,13-tetraazahexadecane-1,16-diamide),HGT4003 (WO 2012/170889, the teachings of which are incorporated hereinby reference in their entirety), ICE (WO 2011/068810, the teachings ofwhich are incorporated herein by reference in their entirety), HGT5000(U.S. Provisional Patent Application No. 61/617,468, the teachings ofwhich are incorporated herein by reference in their entirety) or HGT5001(cis or trans) (Provisional Patent Application No. 61/617,468),aminoalcohol lipidoids such as those disclosed in WO2010/053572, DOTAP(1,2-dioleyl-3-trimethylammonium propane), DOTMA(1,2-di-O-octadecenyl-3-trimethylammonium propane), DLinDMA (Heyes, etal., J. Contr. Rel. 107:276-287(2005)), DLin-KC2-DMA (Semple, et al.,Nature Biotech. 28:172-176 (2010)), C12-200 (Love, et al., Proc. Nat'l.Acad. Sci. 107:1864-1869(2010)). In some embodiments, a cationic lipidis cKK-E12:

Suitable helper lipids include, but are not limited to DSPC(1,2-distearoyl-sn-glycero-3-phosphocholine), DPPC(1,2-dipalmitoyl-sn-glycero-3-phosphocholine), DOPE(1,2-dioleyl-sn-glycero-3-phosphoethanolamine), DPPE(1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine), DMPE(1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine), DOPG(,2-dioleoyl-sn-glycero-3-phospho-(1′-rac-glycerol)), and cholesterol.

PEGylated lipids for use in nanoparticle formulations include, but arenot limited to a poly(ethylene) glycol chain of up to 5 kDa in lengthcovalently attached to a lipid with alkyl chain(s) of C6-C20 length,DMG-PEG2K, PEG-DSG, PEG-DMG, and PEG-ceramides.

In certain embodiments, the lipid nanoparticle carrier comprises one ofthe following lipid formulations:

C12-200, DOPE, cholesterol, DMG-PEG2K;

DODAP, DOPE, cholesterol, DMG-PEG2K;

HGT5000, DOPE, cholesterol, DMG-PEG2K;

HGT5001, DOPE, cholesterol, DMG-PEG2K;

XTC, DSPC, cholesterol, PEG-DMG;

MC3, DSPC, cholesterol, PEG-DMG;

ALNY-100, DSPC, cholesterol, PEG-DSG.

In certain embodiments, the mRNAs of the invention and compositionscomprising those mRNAs may be administered in a local rather thansystemic manner, for example, via injection of the pharmaceuticalcomposition directly into a targeted tissue, preferably in a sustainedrelease formulation. Local delivery can be affected in various ways,depending on the tissue to be targeted. For example, aerosols containingthe mRNAs and compositions of the invention can be inhaled (for nasal,tracheal, or bronchial delivery); mRNAs and compositions of theinvention can be injected into the site of injury, diseasemanifestation, or pain, for example; compositions can be provided inlozenges for oral, tracheal, or esophageal application; can be suppliedin liquid, tablet or capsule form for administration to the stomach orintestines, can be supplied in suppository form for rectal or vaginalapplication; or can even be delivered to the eye by use of creams,drops, or even injection.

Also contemplated herein are lyophilized pharmaceutical compositionscomprising one or more of the liposomal nanoparticles disclosed hereinand related methods for the use of such lyophilized compositions asdisclosed for example, in International Patent Publication WO2012/170889, the teachings of which are incorporated herein by referencein their entirety. For example, lyophilized mRNA and compositions of theinvention may be reconstituted prior to administration or can bereconstituted in vivo. For example, a lyophilized mRNA and/orcomposition can be formulated in an appropriate dosage form (e.g., anintradermal dosage form such as a disk, rod or membrane) andadministered such that the dosage form is rehydrated over time in vivoby the individual's bodily fluids.

In certain embodiments, methods of treating a subject comprisingadministering an mRNA or composition of the invention are alsocontemplated. For example, certain embodiments of the invention providemethods of treating or preventing conditions in which production of aparticular protein and/or utilization of a particular protein isinadequate or compromised. In some embodiments, the present inventionprovides methods of modulating (e.g., increasing, improving or otherwiseenhancing) the translational efficiency of one or more mRNAs in a targetcell. As used herein, the phrase “translational efficiency” refers tothe extent to which an mRNA is translated and the encoded therapeuticprotein is produced.

In certain embodiments, an mRNA molecule of the invention or compositioncomprising such mRNA is administered to a patient.

In some embodiments, an mRNA molecule of the invention or compositioncomprising such mRNA is used for protein production in an in vitro or invivo system. A suitable in vitro system my be an in vitro cell freesystem or an in vitro cell based system. A suitable in vivo system maybe any living organism such as a non-human animal (e.g., rat, mouse,pig, dog, chicken, sheep, non-human primate, etc.) or human.

EXAMPLES

The following specific examples are to be construed as merelyillustrative, and not limiting of the scope of the disclosure. Withoutfurther elaboration, it is believed that one skilled in the art can,based on the description herein, utilize the present invention to itsfullest extent.

Example 1: Synthesis and Expression of mRNA Incorporating4′-Thio-Substituted Furanose Ring

An mRNA which encodes a protein is synthesized. The mRNA contains atleast one 4′-thio-substituted furanose ring. The mRNA is formulated intoa pharmaceutical composition and administered to a subject. The mRNA mayexhibit a longer half-life and result in a greater amount of synthesisof the protein encoded by the mRNA than a control mRNA which does notcontain a 4′-thio-substituted furanose ring.

I. Formulation Experimental Details:

I-a. Messenger RNA Material

Firefly Luciferase (FFL), human erythropoietin (EPO) and humanalpha-galactosidase (GLA) are synthesized by in vitro transcription froma plasmid DNA template encoding the gene, which is followed by theaddition of a 5′ cap structure (Cap1) (Fechter and Brownlee, J. Gen.Virology 86:1239-1249(2005)) and a 3′ poly(A) tail of approximately 200nucleotides in length as determined by gel electrophoresis. 5′ and 3′untranslated regions present in each mRNA product are represented as Xand Y, respectively and defined as stated (vide infra).

Human erythropoietin (EPO) mRNA (SEQ ID NO: 1):X₁AUGGGGGUGCACGAAUGUCCUGCCUGGCUGUGGCUUCUCCUGUCCCUGCUGUCGCUCCCUCUGGGCCUCCCAGUCCUGGGCGCCCCACCACGCCUCAUCUGUGACAGCCGAGUCCUGGAGAGGUACCUCUUGGAGGCCAAGGAGGCCGAGAAUAUCACGACGGGCUGUGCUGAACACUGCAGCUUGAAUGAGAAUAUCACUGUCCCAGACACCAAAGUUAAUUUCUAUGCCUGGAAGAGGAUGGAGGUCGGGCAGCAGGCCGUAGAAGUCUGGCAGGGCCUGGCCCUGCUGUCGGAAGCUGUCCUGCGGGGCCAGGCCCUGUUGGUCAACUCUUCCCAGCCGUGGGAGCCCCUGCAGCUGCAUGUGGAUAAAGCCGUCAGUGGCCUUCGCAGCCUCACCACUCUGCUUCGGGCUCUGGGAGCCCAGAAGGAAGCCAUCUCCCCUCCAGAUGCGGCCUCAGCUGCUCCACUCCGAACAAUCACUGCUGACACUUUCCGCAAACUCUUCCGAGUCUACUCCAAUUUCCUCCGGGGAAAGCUGAAGCUGUACACAGGGGAGGCCUGCAGGACAGGGGACAGAUGAY₁Human alpha-galactosidase (GLA) mRNA (SEQ ID NO: 2):X₁AUGCAGCUGAGGAACCCAGAACUACAUCUGGGCUGCGCGCUUGCGCUUCGCUUCCUGGCCCUCGUUUCCUGGGACAUCCCUGGGGCUAGAGCACUGGACAAUGGAUUGGCAAGGACGCCUACCAUGGGCUGGCUGCACUGGGAGCGCUUCAUGUGCAACCUUGACUGCCAGGAAGAGCCAGAUUCCUGCAUCAGUGAGAAGCUCUUCAUGGAGAUGGCAGAGCUCAUGGUCUCAGAAGGCUGGAAGGAUGCAGGUUAUGAGUACCUCUGCAUUGAUGACUGUUGGAUGGCUCCCCAAAGAGAUUCAGAAGGCAGACUUCAGGCAGACCCUCAGCGCUUUCCUCAUGGGAUUCGCCAGCUAGCUAAUUAUGUUCACAGCAAAGGACUGAAGCUAGGGAUUUAUGCAGAUGUUGGAAAUAAAACCUGCGCAGGCUUCCCUGGGAGUUUUGGAUACUACGACAUUGAUGCCCAGACCUUUGCUGACUGGGGAGUAGAUCUGCUAAAAUUUGAUGGUUGUUACUGUGACAGUUUGGAAAAUUUGGCAGAUGGUUAUAAGCACAUGUCCUUGGCCCUGAAUAGGACUGGCAGAAGCAUUGUGUACUCCUGUGAGUGGCCUCUUUAUAUGUGGCCCUUUCAAAAGCCCAAUUAUACAGAAAUCCGACAGUACUGCAAUCACUGGCGAAAUUUUGCUGACAUUGAUGAUUCCUGGAAAAGUAUAAAGAGUAUCUUGGACUGGACAUCUUUUAACCAGGAGAGAAUUGUUGAUGUUGCUGGACCAGGGGGUUGGAAUGACCCAGAUAUGUUAGUGAUUGGCAACUUUGGCCUCAGCUGGAAUCAGCAAGUAACUCAGAUGGCCCUCUGGGCUAUCAUGGCUGCUCCUUUAUUCAUGUCUAAUGACCUCCGACACAUCAGCCCUCAAGCCAAAGCUCUCCUUCAGGAUAAGGACGUAAUUGCCAUCAAUCAGGACCCCUUGGGCAAGCAAGGGUACCAGCUUAGACAGGGAGACAACUUUGAAGUGUGGGAACGACCUCUCUCAGGCUUAGCCUGGGCUGUAGCUAUGAUAAACCGGCAGGAGAUUGGUGGACCUCGCUCUUAUACCAUCGCAGUUGCUUCCCUGGGUAAAGGAGUGGCCUGUAAUCCUGCCUGCUUCAUCACACAGCUCCUCCCUGUGAAAAGGAAGCUAGGGUUCUAUGAAUGGACUJUCAAGGUUAAGAAGUCACACJAAAUCCCACAGGCACUGUUJUUGCUUCAGCUAGAAAAUACAAUGCAGAUGUCAUUAAAAGACUUACUUUAAY₁Codon-Optimized Firefly Luciferase (FFL) mRNA (SEQ ID NO: 3):X₂AUGGAAGAUGCCAAAAACAUUAAGAAGGGCCCAGCGCCAUUCUACCCACUCGAAGACGGGACCGCCGGCGAGCAGCUGCACAAAGCCAUGAAGCGCUACGCCCUGGUGCCCGGCACCAUCGCCUUUACCGACGCACAUAUCGAGGUGGACAUUACCUACGCCGAGUACUUCGAGAUGAGCGUUCGGCUGGCAGAAGCUAUGAAGCGCUAUGGGCUGAAUACAAACCAUCGGAUCGUGGUGUGCAGCGAGAAUAGCUUGCAGUUCUUCAUGCCCGUGUUGGGUGCCCUGUUCAUCGGUGUGGCUGUGGCCCCAGCUAACGACAUCUACAACGAGCGCGAGCUGCUGAACAGCAUGGGCAUCAGCCAGCCCACCGUCGUAUUCGUGAGCAAGAAAGGGCUGCAAAAGAUCCUCAACGUGCAAAAGAAGCUACCGAUCAUACAAAAGAUCAUCAUCAUGGAUAGCAAGACCGACUACCAGGGCUUCCAAAGCAUGUACACCUUCGUGACUUCCCAUUUGCCACCCGGCUUCAACGAGUACGACUUCGUGCCCGAGAGCUUCGACCGGGACAAAACCAUCGCCCUGAUCAUGAACAGUAGUGGCAGUACCGGAUUGCCCAAGGGCGUAGCCCUACCGCACCGCACCGCUUGUGUCCGAUUCAGUCAUGCCCGCGACCCCAUCUUCGGCAACCAGAUCAUCCCCGACACCGCUAUCCUCAGCGUGGUGCCAUUUCACCACGGCUUCGGCAUGUUCACCACGCUGGGCUACUUGAUCUGCGGCUUUCGGGUCGUGCUCAUGUACCGCUUCGAGGAGGAGCUAUUCUUGCGCAGCUUGCAAGACUAUAAGAUUCAAUCUGCCCUGCUGGUGCCCACACUAUUUAGCUUCUUCGCUAAGAGCACUCUCAUCGACAAGUACGACCUAAGCAACUUGCACGAGAUCGCCAGCGGCGGGGCGCCGCUCAGCAAGGAGGUAGGUGAGGCCGUGGCCAAACGCUUCCACCUACCAGGCAUCCGCCAGGGCUACGGCCUGACAGAAACAACCAGCGCCAUUCUGAUCACCCCCGAAGGGGACGACAAGCCUGGCGCAGUAGGCAAGGUGGUGCCCUUCUUCGAGGCUAAGGUGGUGGACUUGGACACCGGUAAGACACUGGGUGUGAACCAGCGCGGCGAGCUGUGCGUCCGUGGCCCCAUGAUCAUGAGCGGCUACGUUAACAACCCCGAGGCUACAAACGCUCUCAUCGACAAGGACGGCUGGCUGCACAGCGGCGACAUCGCCUACUGGGACGAGGACGAGCACUUCUUCAUCGUGGACCGGCUGAAGAGCCUGAUCAAAUACAAGGGCUACCAGGUAGCCCCAGCCGAACUGGAGAGCAUCCUGCUGCAACACCCCAACAUCUUCGACGCCGGGGUCGCCGGCCUGCCCGACGACGAUGCCGGCGAGCUGCCCGCCGCAGUCGUCGUGCUGGAACACGGUAAAACCAUGACCGAGAAGGAGAUCGUGGACUAUGUGGCCAGCCAGGUUACAACCGCCAAGAAGCUGCGCGGUGGUGUUGUGUUCGUGGACGAGGUGCCUAAAGGACUGACCGGCAAGUUGGACGCCCGCAAGAUCCGCGAGAUUCUCAUUAAGGCCAAGAAGGGCGGCAAGAUCGCCG UGUAY₂X₁ (5′ untranslated sequence) (SEQ ID NO: 4):GGACAGAUCGCCUGGAGACGCCAUCCACGCUGUUUUGACCUCCAUAGAAGACACCGGGACCGAUCCAGCCUCCGCGGCCGGGAACGGUGCAUUGGAACGCGGAUUCCCCGUGCCAAGAGUGACUCACCGUCCUUGACACG X₂ (5′ untranslated sequence)(SEQ ID NO: 5): GGGAUCCUACC Y₁ (3′ untranslated sequence)(SEQ ID NO: 6): CGGGUGGCAUCCCUGUGACCCCUCCCCAGUGCCUCUCCUGGCCCUGGAAGUUGCCACUCCAGUGCCCACCAGCCUUGUCCUAAUAAAAUUAAGUUGCAUCY₂ (3′ untranslated sequence) (SEQ ID NO: 7): UUUGAAUU

I-b. Formulation Protocols

Protocol A: Aliquots of 50 mg/mL ethanolic solutions of C12-200, DOPE,Chol and DMG-PEG2K are mixed and diluted with ethanol to 3 mL finalvolume. Separately, an aqueous buffered solution (10 mM citrate/150 mMNaCl, pH 4.5) of GLA mRNA is prepared from a 1 mg/mL stock. The lipidsolution is injected rapidly into the aqueous mRNA solution and shakento yield a final suspension in 20% ethanol. The resulting nanoparticlesuspension is filtered, diafiltrated with 1×PBS (pH 7.4), concentratedand stored at 2-8° C. Final concentration=0.85 mg/mL GLA mRNA(encapsulated). Zave=81.2 nm (Dv(50)=63.2 nm; Dv(90)=104 nm).

Protocol B: Aliquots of 50 mg/mL ethanolic solutions of DODAP, DOPE,cholesterol and DMG-PEG2K is mixed and diluted with ethanol to 3 mLfinal volume. Separately, an aqueous buffered solution (10 mMcitrate/150 mM NaCl, pH 4.5) of EPO mRNA is prepared from a 1 mg/mLstock. The lipid solution is injected rapidly into the aqueous mRNAsolution and shaken to yield a final suspension in 20% ethanol. Theresulting nanoparticle suspension was filtered, diafiltrated with 1×PBS(pH 7.4), concentrated and stored at 2-8° C. Final concentration=1.35mg/mL EPO mRNA (encapsulated). Zave=75.9 nm (Dv(50)=57.3 nm; Dv(90)=92.1nm).

Protocol C: Aliquots of a 2.0 mg/mL aqueous solution PEI (branched, 25kDa) is mixed with aqueous solution of CFTR mRNA (1.0 mg/mL). Theresulting complexed mixture is pipetted up and down several times andput aside for 20 minutes prior to injection. Final concentration=0.60mg/mL CFTR mRNA (encapsulated). Zave=75.9 nm (Dv(50)=57.3 nm;Dv(90)=92.1 nm).

II. Analysis of Modified Messenger RNA Versus Unmodified mRNA:

II-a. Quantification of Modified Base Within Messenger RNA Construct

4′-Thio NTP modified messenger RNA are subjected to RNase I or NucleaseP1 for various periods of time to allow for sufficient degradation. Uponcompletion, the resulting monophosphate nucleotides are degraded furtherwith alkaline phosphatase to provide the respective nucleosides. Thenucleoside mixture is applied to an Amicon spin column (30,000 MWCO) forefficient enzyme removal. The resulting nucleoside solution is analyzedvia HPLC and quantified via peak area comparison with respectiveunmodified nucleoside.

II-b. Stability of 4′-Thio NTP Modified Messenger RNA Construct

4′-Thio NTP modified messenger RNA is subjected to RNase I or NucleaseP1 for a various periods of time to assess resistance to nucleasedegradation. Similarly, 4′-thio NTP modified messenger RNA was treatedwith serum (containing nucleases) over various time periods to assessnuclease degradation. At specified time points, the nuclease reactionsare quenched with inhibitor and the resulting solution is applied to anAmicon spin column (30,000 MWCO) for efficient enzyme removal. Uponcompletion, the retentate is applied to a 1% agarose gel and analyzedfor mRNA construct viability (size, degradation products, etc).Identical experiments are performed on unmodified mRNA and directcomparisons and inferences may be drawn.

II-c. 4′-Thio NTP Modified Messenger RNA Effects on Protein Production

In Vitro Studies: In vitro transfections of 4′-thio NTP modified mRNAand unmodified mRNA are performed using HEK293T cells. Transfections ofone microgram of each mRNA construct are performed in separate wellsusing lipofectamine. Cells are harvested at select time points (eg. 4hour, 8 hour, 24 hour, 48 hour, 72 hour, etc.) and respective proteinproduction are analyzed. For FFL mRNA, cell lysates are analyzed forluciferase production via bioluminescence assays. For EPO and GLA mRNAstudies, cell supernatants are obtained and analyzed for EPO and GLAprotein, respectively, using ELISA-based methods. A comparison ofprotein production over time of unmodified versus 4′-thio NTP modifiedmRNA may be made.

In Vivo Studies: A comparison of protein production over time is madevia injection of 4′thio NTP modified mRNA encapsulated nanoparticles(lipid or polymeric) into wild type mice (CD-1) versus unmodified mRNAdelivered in identical fashion. Serum and organs were collected atselect time points (e.g. 6 hr, 12 hr, 24 hr, 48 hr, 72 hr, etc.) andrespective protein levels are monitored. For FFL mRNA, liver homogenatesare analyzed for luciferase production via bioluminescence assays. ForEPO and GLA mRNA studies, mouse sera are obtained and analyzed for EPOand GLA protein, respectively, using ELISA-based methods. A comparisonof protein production over time of unmodified versus 4′-thio NTPmodified mRNA is made.

Similarly, unencapsulated (naked) 4′thio NTP modified mRNA andunmodified mRNA are injected via either intravenous, subcutaneous orintratracheal administration and identical analyses may be performed asdescribed above to assess differences of stability and proteinproduction.

III. Analysis of FFL, EPO and GLA Protein Produced Via AdministeredNaked Modified mRNA or mRNA-Loaded Nanoparticles:

III-a. Injection Protocol

All studies are performed using male CD-1 mice of approximately 6-8weeks of age at the beginning of each experiment. Samples are introducedby a single bolus tail-vein injection of an equivalent total dose of30-200 micrograms of unencapsulated or encapsulated FFL, EPO or GLA mRNA(modified or unmodified). Mice are sacrificed and perfused with salineat the designated time points.

III-b. Isolation of Organ Tissues for Analysis

The liver and spleen of each mouse is harvested, apportioned into threeparts, and stored in either 10% neutral buffered formalin or snap-frozenand stored at −80° C. for analysis.

III-c. Isolation of Serum for Analysis

All animals are euthanized by CO₂ asphyxiation 48 hours post doseadministration (±5%) followed by thoracotomy and terminal cardiac bloodcollection. Whole blood (maximal obtainable volume) is collected viacardiac puncture on euthanized animals into serum separator tubes,allowed to clot at room temperature for at least 30 minutes, centrifugedat 22° C.±5° C. at 9300 g for 10 minutes, and the serum is extracted.For interim blood collections, approximately 40-50 μL of whole blood iscollected via facial vein puncture or tail snip. Samples collected fromnon-treatment animals are used as a baseline GLA levels for comparisonto study animals.

III-d. Enzyme-Linked Immunosorbent Assay (ELISA) Analysis

EPO ELISA: Quantification of EPO protein is performed followingprocedures reported for human EPO ELISA kit (Quantikine IVD, R&DSystems, Catalog #Dep-00). Positive controls that may be employedconsist of ultrapure and tissue culture grade recombinant humanerythropoietin protein (R&D Systems, Catalog #286-EP and 287-TC,respectively). Blood samples are taken at designated time points andprocessed as described above. Detection is monitored via absorption (450nm) on a Molecular Device Flex Station instrument.

GLA ELISA: Standard ELISA procedures are followed employing sheepanti-REPLAGAL® G-188 IgG as the capture antibody with rabbitanti-REPLAGAL® TK-88 IgG as the secondary (detection) antibody (ShireHuman Genetic Therapies). Horseradish peroxidase (HRP)-conjugated goatanti-rabbit IgG is used for activation of the3,3′,5,5′-tetramethylbenzidine (TMB) substrate solution. The reaction isquenched using 2N H₂SO₄ after 20 minutes. Detection is monitored viaabsorption (450 nm) on a Molecular Device Flex Station instrument.Untreated mouse serum and human REPLAGAL® protein is used as negativeand positive controls, respectively.

III-e. Bioluminescence Analysis

Luciferase Assay: The bioluminescence assay is conducted using a PromegaLuciferase Assay System (Item #E1500). The Luciferase Assay Reagent isprepared by adding 10 mL of Luciferase Assay Buffer to Luciferase AssaySubstrate and mix via vortex. Approximately 20 uL of homogenate samplesare loaded onto a 96-well plate followed by 20 uL of plate control toeach sample. Separately, 120 uL of Luciferase Assay Reagent (prepared asdescribed above) is added to each well of a 96-well flat bottomed plate.Each plate is then inserted into the appropriate chambers using aMolecular Device Flex Station instrument and measure the luminescence(measured in relative light units (RLU)).

Example 2. Exemplary Liposome Formulations for Delivery and Expressionof mRNA with 4′-Thio Modifications

This example provides exemplary liposome formulations for effectivedelivery and expression of 4′-Thio modified mRNA in vivo.

Lipid Materials

The formulations described herein include a multi-component lipidmixture of varying ratios employing one or more cationic lipids, helperlipids (e.g., non-cationic lipids and/or cholesterol-based lipids) andPEGylated lipids designed to encapsulate various nucleic acid-basedmaterials. Cationic lipids can include (but not exclusively) DOTAP(1,2-dioleyl-3-trimethylammonium propane), DODAP(1,2-dioleyl-3-dimethylammonium propane), DOTMA(1,2-di-O-octadecenyl-3-trimethylammonium propane), DLinDMA (Heyes, J.;Palmer, L.; Bremner, K.; MacLachlan, I. “Cationic lipid saturationinfluences intracellular delivery of encapsulated nucleic acids” J.Contr. Rel. 2005, 107, 276-287), DLin-KC2-DMA (Semple, S. C. et al.“Rational Design of Cationic Lipids for siRNA Delivery” Nature Biotech.2010, 28, 172-176), C12-200 (Love, K. T. et al. “Lipid-like materialsfor low-dose in vivo gene silencing” PNAS 2010, 107, 1864-1869),HGT4003, HGT5000, HGT5001, MC3, cKK-E12(3,6-bis(4-(bis(2-hydroxydodecyl)amino)butyl)piperazine-2,5-dione), ICE,dialkylamino-based, imidazole-based, guanidinium-based, etc. Helperlipids can include (but not exclusively) DSPC(1,2-distearoyl-sn-glycero-3-phosphocholine), DPPC(1,2-dipalmitoyl-sn-glycero-3-phosphocholine), DOPE(1,2-dioleyl-sn-glycero-3-phosphoethanolamine), DPPE(1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine), DMPE(1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine), DOPG(,2-dioleoyl-sn-glycero-3-phospho-(1′-rac-glycerol)), cholesterol, etc.The PEGylated lipids can include (but not exclusively) a poly(ethylene)glycol chain of up to 5 kDa in length covalently attached to a lipidwith alkyl chain(s) of C₆-C₂₀ length.

Polymeric Materials

Further formulations described herein include various charged, polymericmaterials which can include (but not exclusively) branchedpolyethyleneimine (PEI) (25 kDa) (Sigma #408727), protamine, PEGylatedprotamine, PLL, PEGylated PLL, etc.

mRNA Materials

Firefly Luciferase (FFL), human erythropoietin (EPO) and humanalpha-galactosidase (GLA) were synthesized by in vitro transcriptionfrom a plasmid DNA template encoding the gene, which was followed by theaddition of a 5′ cap structure (Cap1) (Fechter, P.; Brownlee, G. G.“Recognition of mRNA cap structures by viral and cellular proteins” J.Gen. Virology 2005, 86, 1239-1249) and a 3′ poly(A) tail ofapproximately 200 nucleotides in length as determined by gelelectrophoresis. 5′ and 3′ untranslated regions present in each mRNAproduct are represented as X and Y, respectively and defined as stated(vide infra).

Exemplary mRNA sequences of human erythropoietin (EPO), humanalpha-galactosidase (GLA) and Codon-Optimized Firefly Luciferase (FFL)are depicted in SEQ ID No. 1, 2, and 3, respectively. Exemplary 5′ and3′ UTR sequences are described in SEQ ID Nos. 4, 5, 6, and 7.

Exemplary Formulation Protocols

A. C12-200 and GLA

Aliquots of 50 mg/mL ethanolic solutions of C12-200, DOPE, cholesteroland DMG-PEG2K were mixed and diluted with ethanol to 3 mL final volume.Separately, an aqueous buffered solution (10 mM citrate/150 mM NaCl, pH4.5) of GLA mRNA was prepared from a 1 mg/mL stock. The lipid solutionwas injected rapidly into the aqueous mRNA solution and shaken to yielda final suspension in 20% ethanol. The resulting nanoparticle suspensionwas filtered, diafiltrated with 1×PBS (pH 7.4), concentrated, and storedat 2-8° C. Final concentration=0.85 mg/mL GLA mRNA (encapsulated).Z_(ave)=81.2 nm (Dv₍₅₀₎=63.2 nm; Dv₍₉₀₎=104 nm).

B. DODAP and EPO

Aliquots of 50 mg/mL ethanolic solutions of DODAP, DOPE, cholesterol andDMG-PEG2K were mixed and diluted with ethanol to 3 mL final volume.Separately, an aqueous buffered solution (10 mM citrate/150 mM NaCl, pH4.5) of EPO mRNA was prepared from a 1 mg/mL stock. The lipid solutionwas injected rapidly into the aqueous mRNA solution and shaken to yielda final suspension in 20% ethanol. The resulting nanoparticle suspensionwas filtered, diafiltrated with 1×PBS (pH 7.4), concentrated, and storedat 2-8° C. Final concentration=1.35 mg/mL EPO mRNA (encapsulated).Z_(ave)=75.9 nm (Dv₍₅₀₎=57.3 nm; Dv₍₉₀₎=92.1 nm).

C. PEI and CFTR

Aliquots of a 2.0 mg/mL aqueous solution PEI (branched, 25 kDa) weremixed with aqueous solution of CFTR mRNA (1.0 mg/mL). The resultingcomplexed mixture was pipetted up and down several times and put asidefor 20 minutes prior to injection. Final concentration=0.60 mg/mL CFTRmRNA (encapsulated). Z_(ave)=75.9 nm (Dv₍₅₀₎=57.3 nm; Dv₍₉₀₎=92.1 nm).

Example 3. Analysis of In Vivo Stability and Protein Production ofModified mRNA Versus Unmodified mRNA

This example illustrates exemplary methods for analyzing stability ofmodified mRNA and protein expression in various target tissues in vivo.

Quantification of Modified Base within mRNA Construct

4′-Thio NTP Modified mRNA were subjected to RNase I or Nuclease P1 forvarious periods of time to allow for sufficient degradation. Uponcompletion, the resulting monophosphate nucleotides were degradedfurther with alkaline phosphatase to provide the respective nucleosides.The nucleoside mixture was applied to an Amicon spin column (30,000MWCO) for efficient enzyme removal. The resulting nucleoside solutionwas analyzed via HPLC and quantified via peak area comparison withrespective unmodified nucleoside.

Stability of 4′-Thio NTP Modified mRNA Construct

4′-Thio NTP Modified mRNA were subjected to RNase I or Nuclease P1 for avarious periods of time to assess resistance to nuclease degradation. Atspecified time points, the nuclease reactions were quenched withinhibitor and the resulting solution was applied to an Amicon spincolumn (30,000 MWCO) for efficient enzyme removal. Upon completion, theretentate was applied to a 1% agarose gel and analyzed for mRNAconstruct viability (size, degradation products, etc). Identicalexperiments were performed on unmodified mRNA and direct comparisons andinferences were drawn.

4′-Thio NTP Modified mRNA Effects on Protein Production

In Vitro Studies:

In vitro transfections of 4′-thio NTP modified mRNA and unmodified mRNAwere performed using HEK293T cells. Transfections of one microgram ofeach mRNA construct were performed in separate wells usinglipofectamine. Cells were harvested at select time points (e.g. 4 hour,8 hour, 32 hour, 48 hour, 56 hour, 80 hour, etc.) and respective proteinproduction was analyzed. For FFL mRNA, cell lysates were analyzed forluciferase production via bioluminescence assays. For EPO and GLA mRNAstudies, cell supernatants were obtained and analyzed for EPO and GLAprotein, respectively, using ELISA-based methods. A comparison ofprotein production over time of unmodified versus 4′-thio NTP modifiedmRNA was made. Exemplary results are shown in FIG. 2 .

In Vivo Studies:

A comparison of protein production over time was made via injection of4′thio NTP modified mRNA encapsulated nanoparticles (lipid or polymeric)into wild type mice (CD-1) versus unmodified mRNA delivered in anidentical fashion. Serum and organs were collected at select time points(eg. 6 hr, 12 hr, 24 hr, 48 hr, 72 hr, etc.) and respective proteinlevels were monitored. For FFL mRNA, liver homogenates were analyzed forluciferase production via bioluminescence assays. For EPO and GLA mRNAstudies, mouse sera were obtained and analyzed for EPO and GLA protein,respectively, using ELISA-based methods. A comparison of proteinproduction over time of unmodified versus 4′-thio NTP modified mRNA wasmade.

Similarly, unencapsulated (naked) 4′thio NTP modified mRNA andunmodified mRNA were injected via either intravenous, subcutaneous orintratracheal administration and identical analyses were performed toassess differences in stability and protein production.

Example 4. Analysis of FFL, EPO and GLA Protein Production AfterAdministration of Naked Modified mRNA or mRNA-Loaded Nanoparticles

This example describes the protocol for analyzing exemplary proteinproduction after administering either naked, modified, mRNA ormRNA-loaded nanoparticles and demonstrates mRNA stability and proteinproduction for 4′-thio modified mRNA compared to unmodified mRNA.

All studies were performed using male CD-1 mice of approximately 6-8weeks of age at the beginning of each experiment. Samples wereintroduced by a single bolus tail-vein injection of an equivalent totaldose of 30-200 micrograms of unencapsulated or encapsulated FFL, EPO orGLA mRNA (modified or unmodified). Mice were sacrificed and perfusedwith saline at the designated time points.

The liver and spleen of each mouse was harvested, apportioned into threeparts, and stored in either 10% neutral buffered formalin or snap-frozenand stored at −80° C. for analysis.

All animals were euthanized by CO₂ asphyxiation 48 hours post doseadministration (±5%) followed by thoracotomy and terminal cardiac bloodcollection. Whole blood (maximal obtainable volume) was collected viacardiac puncture from euthanized animals into serum separator tubes,allowed to clot at room temperature for at least 30 minutes, centrifugedat 22° C.±5° C. at 9300 g for 10 minutes, after which time serum wasextracted. For interim blood collections, approximately 40-50 μL ofwhole blood was collected via facial vein puncture or tail snip. Samplescollected from non-treatment animals were used for baseline GLA levelsfor comparison to study animals.

Enzyme-Linked Immunosorbent Assay (ELISA) Analysis

Quantification of EPO protein was performed following proceduresreported for human EPO ELISA kit (Quantikine IVD, R&D Systems, Catalog#Dep-00). Positive controls employed consisted of ultrapure and tissueculture grade recombinant human erythropoietin protein (R&D Systems,Catalog #286-EP and 287-TC, respectively). Blood samples were taken atdesignated time points and processed as described above. Detection wasmonitored via absorption (450 nm) on a Molecular Device Flex Stationinstrument.

For analysis of GLA protein, standard ELISA procedures were followedemploying sheep anti-Replagal G-188 IgG as the capture antibody withrabbit anti-Replagal IgG as the secondary (detection) antibody.Horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG was usedfor activation of the 3,3′,5,5′-tetramethylbenzidine (TMB) substratesolution. The reaction was quenched using 2N H₂SO₄ after 20 minutes.Detection was monitored via absorption (450 nm) on a Molecular DeviceFlex Station instrument. Untreated mouse serum and human Replagal®protein were used as negative and positive controls, respectively.Bioluminescence Analysis

The bioluminescence assay was conducted using a Promega Luciferase AssaySystem (Item #E1500). The Luciferase Assay Reagent was prepared byadding 10 mL of Luciferase Assay Buffer to Luciferase Assay Substrateand mixed via vortex. 20 uL of homogenate samples were loaded onto a96-well plate followed by 20 uL of plate control to each sample.Separately, 120 uL of Luciferase Assay Reagent (prepared as describedabove) was loaded into each well of a 96-well flat bottomed plate. Eachplate was then inserted into the appropriate chambers using a MolecularDevice Flex Station instrument and the luminescence was measured inrelative light units (RLU).

Exemplary Results

The production of FFL protein via transfection of 4′-thio modified orunmodified FFL mRNA was tested in HEK 293T cells. FIG. 2 represents theweighted relative fluorescence units (RLU) scores taken 4 hours, 8hours, 32 hours, 56 hours and 80 hours after transfection. At each timepoint, cells transfected with either 25% 4′-thio uridine (25% 4′-S-U) or100% 4′-thio uridine (100% 4′-S-U) FFL mRNA had higher weighted RLUscores than cells transfected with either SNIM® FFL or commercial FFL.

The stability of 4′-thio modified and unmodified FFL mRNA over time wasalso tested. Three micrograms of mRNA were exposed to mouse serum andmonitored over the course of one hour. As can be seen in FIG. 3 ,compared to SNIM® FFL and commercial FFL mRNA, 4′-thio modified mRNA,particularly 100% 4′-thio uridine (100% 4′-S-U) FFL mRNA, appears to bemore stable over time.

The production of FFL protein via transfection of 4′-thio modified orunmodified FFL mRNA was tested in wild-type mice. A 1.0 mg/kg dose ofC12-200-loaded lipid nanoparticles was administered intravenously andanimals were sacrificed and their livers were removed for analysis, asdescribed above. FIG. 4 represents RLU/mg Total Protein scores taken sixhours post-administration. Livers from mice treated with 25% 4′-thiouridine (25% 4′-S-U) and 100% 4′-thio uridine (100% 4′-S-U) had higherRLU/mg scores than the livers from mice treated with unmodified mRNA.

Among other things, the exemplary results described herein demonstratedthat a provided mRNA comprising 4′-thio-modified nucleotide can besuccessfully synthesized, have increased stability, and can be usedsuccessfully to produce protein in cells.

Example 5. Exemplary Syntheses of 4′-Thio-Modified Nucleotides

Synthetic Procedures:

Preparation of Intermediates:

Synthesis of 2,3-O-isopropylidene-D-ribonic acid-1,4-lactone

A solution of D-ribonic acid-1,4-lactone (270.0 g, 1.823 mol) andsulphuric acid (18.0 g, 0.182 mol, 0.1 equiv.) in acetone (2.79 L) wasstirred at room temperature for 3 days. The reaction mixture wasquenched by the addition of solid sodium bicarbonate (˜450 g), filteredand the filtrate evaporated. The residue was partitioned between ethylacetate and water. The aqueous layer was extracted with ethyl acetate;the combined organic layers were dried over magnesium sulphate, filteredand concentrated under reduced pressure to give the desired product as awhite solid (318.8 g, 93%). ¹H NMR (300 MHz, CDCl₃) δ 4.83 (d, J=5.5 Hz,1H), 4.77 (d, J=5.5 Hz, 1H), 4.64-4.62 (m, 1H), 3.99 (ddd, J=2.3, 5.5and 12.4 Hz, 1H), 3.81 (ddd, J=2.3, 5.5 and 12.4 Hz, 1H), 2.67 (t, J=5.5Hz, 1H), 1.46 (s, 3H), 1.37 (s, 3H).

Synthesis of 5-O-methanesulfonyl-2,3-O-isopropylidene-D-ribonicacid-1,4-lactone

Methanesulfonyl chloride (116.0 g, 1.014 mol, 1.2 equiv.) was addeddropwise at 0° C. to a solution of 2,3-O-isopropylidene-D-ribonicacid-1,4-lactone (159.0 g, 0.845 mol) and triethylamine (128.0 g, 1.267mol) in dichloromethane (2.43 L). The reaction mixture was stirred atroom temperature for 1 h. and was then diluted with dichloromethane andwashed with water, sat. aq. NaHCO₃ and brine. The organic layer wasdried over magnesium sulphate, filtered and concentrated under reducedpressure to give 5-O-methanesulfonyl-2,3-O-isopropylidene-D-ribonicacid-1,4-lactone as an orange oil (231.0 g, ca. 100%). ¹H NMR (300 MHz,CDCl₃) δ 4.82-4.77 (m, 3H), 4.49-4.41 (m, 2H), 3.04 (s, 3H), 1.47 (s,3H), 1.38 (s, 3H).

Synthesis of 2,3-O-isopropylidene-L-lyxonic acid-1,4-lactone

Potassium hydroxide (137.0 g, 2.45 mol) in water (1.1 L) was added to5-O-methanesulfonyl-2,3-O-isopropylidene-D-ribonic acid-1,4-lactone(225.0 g, 0.85 mol) and stirred at room temperature for 18 h. Thereaction mixture was acidified to pH 3 with 2M aq. HCl (using a pHmeter) then evaporated. The residue was heated to reflux in acetone andthe acetone decanted (×3). The combined extracts were dried overmagnesium sulphate, filtered and concentrated under reduced pressure togive 2,3-O-isopropylidene-L-lyxonic acid-1,4-lactone as a pale yellowsolid (115.4 g, 73%). ¹H NMR (300 MHz, CDCl₃) δ 4.89-4.84 (m, 2H),4.63-4.59 (m, 1H), 4.08-3.91 (m, 2H), 1.46 (s, 3H), 1.38 (s, 3H).

Synthesis of 5-O-tert-Butyldimethylsilyl-2,3-O-isopropylidene-L-lyxonicacid-1,4-lactone

To a solution of 2,3-O-isopropylidene-L-lyxonic acid-1,4-lactone (5.0 g,0.027 mol) in dichloromethane (85.0 ml) was added imidazole (2.2 g, 32mmol) followed by tert-butyldimethyllsilyl chloride (4.4 g, 29 mmol, 1.1equiv.) and the reaction mixture stirred at room temperature for 1.5 h.The reaction mixture was diluted with dichloromethane, washed with sat.aq. NaHCO₃, brine, dried over magnesium sulphate, filtered andconcentrated under reduced pressure to give5-O-tert-butyldimethylsilyl-2,3-O-isopropylidene-L-lyxonicacid-1,4-lactone as a pale yellow oil (7.3 g, 89%). ¹H NMR (300 MHz,CDCl₃) δ 4.79 (s, 2H), 4.54-4.49 (m, 1H), 4.00-3.86 (m, 2H), 1.45 (s,3H), 1.38 (s, 3H), 0.89 (s, 9H), 0.08 (s, 6H).

Synthesis of 5-O-tert-Butyldimethylsilyl-2,3-O-isopropylidene-L-lyxitol

To a solution of5-O-tert-butyldimethylsilyl-2,3-O-isopropylidene-L-lyxonicacid-1,4-lactone (7.2 g, 24 mmol) in tetrahydrofuran (63 ml) andmethanol (13 ml) was added sodium borohydride (1.4 g, 0.036 mol, 1.5equiv.) portion-wise at room temperature. The reaction mixture wasstirred for 1 h at room temperature and then concentrated under reducedpressure. The residue was partitioned between ethyl acetate and 1M aq.citric acid. The organic layer was washed with 1M aq. citric acid,brine, dried over magnesium sulphate, filtered and concentrated underreduced pressure to give5-O-tert-butyldimethylsilyl-2,3-O-isopropylidene-L-lyxitol as acolourless oil which crystallised upon standing (6.3 g, 86%). ¹H NMR(300 MHz, CDCl₃) δ 4.26-4.20 (m, 2H), 3.84-3.59 (m, 5H), 1.51 (s, 3H),1.37 (s, 3H), 0.89 (s, 9H), 0.07 (s, 6H).

Synthesis of5-O-tert-Butyldimethylsilyl-2,3-O-isopropylidene-1,4-di-O-methanesulfonyl-L-lyxitol

Methanesulfonyl chloride (15.2 ml, 0.196 mol, 10 equiv.) was addeddrop-wise at <10° C. to pyridine (15.8 ml, 0.196 mol, 10 equiv.). Asolution of 5-O-tert-butyldimethylsilyl-2,3-O-isopropylidene-L-lyxitol(6 g, 0.0196 mol) in dichloromethane (16 ml) was added drop-wise at <10°C. The reaction mixture was stirred at rt for 2 h. The cooling bath wasreplaced and the excess of methanesulfonyl chloride was hydrolyzed byaddition of ice. The reaction mixture was then poured in water (300 mL)and extracted with Et₂O (3×50 mL). The combined organic layers werewashed with 1M aq. citric acid, sat. aq. NaHCO₃ and brine, dried(MgSO₄), filtered and concentrated under reduced pressure. The crudeproduct was purified by flash chromatography on silica gel with 1:6ethyl acetate/heptane to give5-O-tert-butyldimethylsilyl-2,3-O-isopropylidene-1,4-di-O-methanesulfonyl-L-lyxitolas yellow oil (8 g, 91%). ¹H NMR (300 MHz, CDCl₃) δ 4.76-4.69 (m, 1H),4.46-4.34 (m, 4H), 3.95 (dd, J=5.5 and 11.0 Hz, 1H), 3.82 (dd, J=6.0 and11.0 Hz, 1H), 3.11 (s, 3H), 3.07 (s, 3H), 1.51 (s, 3H), 1.37 (s, 3H),0.89 (s, 9H), 0.09 (s, 6H).

Synthesis oftert-butyl(((3aS,4R,6aR)-2,2-dimethyltetrahydrothieno[3,4-d][1,3]dioxol-4-yl)methoxy)dimethylsilane

To a solution of5-O-tert-butyldimethylsilyl-2,3-O-isopropylidene-1,4-di-O-methanesulfonyl-L-lyxitol(25.1 g, 0.056 mol) in dimethylformamide (250 ml) was added Na₂S·9H₂O(16.1 g, 0.067 mol, 1.2 eq.) and the reaction mixture was heated at 80°C. for 3 h. The reaction mixture was cooled to room temperature,partitioned between water and ethyl acetate. The layers were separatedand the aqueous layer was extracted with ethyl acetate. The combinedorganic layers were dried (MgSO₄), filtered and concentrated underreduced pressure. The crude product was purified by flash chromatographyon silica gel with ethyl acetate/heptane (10:1) to givetert-butyl(((3aS,4R,6aR)-2,2-dimethyltetrahydrothieno[3,4-d][1,3]dioxol-4-yl)methoxy)-dimethyl-silane(10.3 g, 60%). ¹H NMR (300 MHz, CDCl₃) δ 4.89 (dt, J=1.4 and 4.6 Hz,1H), 4.79 (d, J=6.0 Hz, 1H), 3.80 (dd, J=5.0 and 10.5 Hz, 1H), 3.60 (dd,J=6.4 and 10.6 Hz, 1H), 3.33 (t, J=5.0 Hz, 1H), 3.16 (dd, J=5.0 and 12.4Hz, 1H), 2.85 (dd, J=0.9 and 12.8 Hz, 1H), 1.52 (s, 3H), 1.32 (s, 3H),0.89 (s, 9H), 0.06 (s, 6H).

Synthesis of(3aS,4R,5R,6aR)-4-(((tert-butyldimethylsilyl)oxy)methyl)-2,2-dimethyltetrahydrothieno[3,4-d][1,3]dioxole5-oxide

A solution of ca 70% m-chloroperbenzoic acid (16 g, 66 mmol) indichloromethane (150 ml) was dried over magnesium sulphate and filtered.After washing with dichloromethane (50 ml), the combined filtrate wasadded drop-wise to a solution oftert-butyl(((3aS,4R,6aR)-2,2-dimethyltetrahydrothieno[3,4-d][1,3]dioxol-4-yl)methoxy)dimethylsilane(20 g, 66 mmol) in dichloromethane (400 ml) at −78° C. After stirringfor an hour at −78° C., the reaction was quenched with saturated sodiumbicarbonate solution and diluted with dichloromethane. The layers wereseparated and the organic washed with brine, dried over magnesiumsulphate, filtered and concentrated. The residue obtained was purifiedby flash chromatography (silica gel/dichloromethane:diethyl ether (30:1)to give(3aS,4R,5S,6aR)-4-(((tert-butyldimethylsilyl)oxy)methyl)-2,2-dimethyltetrahydrothieno[3,4-d][1,3]dioxole5-oxide (8.8 g, 42%) and(3aS,4R,5R,6aR)-4-(((tert-butyldimethylsilyl)oxy)methyl)-2,2-dimethyltetra-hydrothieno[3,4-d][1,3]dioxole5-oxide (7.3 g, 35%).

Synthesis of Nucleoside Intermediates:

1-((3aR,4R,6R,6aS)-6-(hydroxymethyl)-2,2-dimethyltetrahydrothieno[3,4-d][1,3]dioxol-4-yl)pyrimidine-2,4(1H,3H)-dione

Synthesis of1-((3aR,4R,6R,6aS)-6-(((tert-butyldimethylsilyl)oxy)methyl)-2,2-dimethyl-tetrahydrothieno[3,4-d][1,3]dioxol-4-yl)pyrimidine-2,4(1H,3H)-dione

To a suspension of uracil (1.40 g, 12.5 mmol) in toluene (62 ml) wasadded triethylamine (3.5 ml, 2.53 g, 25 mmol) and trimethylsilyltrifluoromethanesulfonate (9.01 ml, 11.1 g, 50 mmol). After stirring foran hour at room temperature, dichloromethane (34 ml) was added to thebi-phasic mixture to give a solution; this was then added drop-wise to asolution of(3aS,4R,5R,6aR)-4-(((tert-butyldimethylsilyl)oxy)methyl)-2,2-dimethyltetrahydrothieno[3,4-d][1,3]dioxole5-oxide (2.0 g, 6.25 mmol) in dichloromethane (34 ml) and thentriethylamine (3.5 ml, 25 mmol) was added. After stirring for 90 minutesat room temperature, the reaction was quenched with ice and then dilutedwith ethyl acetate. The layers were separated and the organic washedwith saturated sodium bicarbonate solution (×2) and then brine. Afterdrying over magnesium sulphate, concentration under reduced pressuregave crude product which was purified by flash chromatography (silicagel, ethyl acetate:dichloromethane 1:5) to give1-((3aR,4R,6R,6aS)-6-(((tert-butyldimethylsilyl)oxy)methyl)-2,2-dimethyl-tetrahydrothieno[3,4-d][1,3]dioxol-4-yl)pyrimidine-2,4(1H,3H)-dione(1.55 g, 60%). ¹H NMR (300 MHz, CDCl₃) δ 8.43 (s, 1H), 7.96 (d, J=8.3Hz, 1H), 6.12 (d, J=2.3 Hz, 1H), 5.74 (dd, J=1.9 and 7.8 Hz, 1H), 4.71(m, 2H), 3.89 (m, 2H), 3.74 (m, 1H), 1.60 (s, 3H), 1.32 (s, 3H), 0.92(s, 9H), 0.11 (s, 3H), 0.10 (s, 3H).

Synthesis of1-((3aR,4R,6R,6aS)-6-(hydroxymethyl)-2,2-dimethyltetrahydrothieno[3,4-d][1,3]dioxol-4-yl)pyrimidine-2,4(1H,3H)-dione

A solution of1-((3aR,4R,6R,6aS)-6-(((tert-butyldimethylsilyl)oxy)methyl)-2,2-dimethyl-tetrahydrothieno[3,4-d][1,3]dioxol-4-yl)pyrimidine-2,4(1H,3H)-dione(3.70 g, 8.92 mmol) in tetrahydrofuran (85 ml) was cooled in an ice bathunder argon; a solution of 1M tetrabutylammonium fluoride intetrahydrofuran (10.7 ml, 10.7 mmol) was added and the mixture stirredfor 2 hrs at room temperature. The crude product was collected byfiltration, washed with tetrahydrofuran and purified by flashchromatography (silica gel; methanol:dichloromethane 1:30) to give1-((3aR,4R,6R,6aS)-6-(hydroxymethyl)-2,2-dimethyltetrahydrothieno[3,4-d][1,3]dioxol-4-yl)pyrimidine-2,4(1H,3H)-dione(2.30 g, 86%). ¹H NMR (300 MHz, CDCl₃) δ 8.97 (brs, 1H), 7.76 (d, J=8.3Hz, 1H), 5.93 (s, 1H), 5.76 (d, J=8.3 Hz), 4.91 (s, 2H), 3.96 (dd, J=4.6and 11.0 Hz, 1H), 3.89 (dd, J=4.6 and 11.0 Hz, 1H), 3.79 (t, J=4.6 Hz,1H), 2.64 (brs, 1H), 1.59 (s, 3H), 1.33 (s, 3H).

4-Amino-1-((3aR,4R,6R,6aS)-6-(hydroxymethyl)-2,2-dimethyltetrahydrothieno[3,4-d][1,3]dioxol-4-yl)-5-methylpyrimidin-2(1H)-one

Using a procedure analogous to the following, but substituting cytosinefor thymine,4-amino-1-((3aR,4R,6R,6aS)-6-(hydroxymethyl)-2,2-dimethyltetrahydrothieno[3,4-d][1,3]dioxol-4-yl)pyrimidin-2(1H)-one (13.1) can be synthesized.

Synthesis of1-((3aR,4R,6R,6aS)-6-(((tert-butyldimethylsilyl)oxy)methyl)-2,2-dimethyltetrahydrothieno[3,4-d][1,3]dioxol-4-yl)-5-methylpyrimidine-2,4(1H,3H)-dione

To a suspension of thymine (2.60 g, 20.6 mmol) in toluene (112 ml) wasadded triethylamine (4.17 g, 41.2 mmol) and trimethylsilyltrifluoromethanesulfonate (18.3 g, 82.5 mmol). After stirring for anhour at room temperature, dichloromethane (34 ml) was added to thebi-phasic mixture to give a solution; this was then added drop-wise to asolution of(3aS,4R,5R,6aR)-4-(((tert-butyldimethylsilyl)oxy)methyl)-2,2-dimethyltetrahydrothieno[3,4-d][1,3]dioxole5-oxide (3.30 g, 10.3 mmol) in dichloromethane (56 ml) and thentriethylamine (4.17 g, 41.2 mmol) was added. After stirring for 60 minat room temperature, the reaction was quenched with ice and then dilutedwith ethyl acetate. The layers were separated and the organic washedwith saturated sodium bicarbonate solution (×2) and then brine. Afterdrying over magnesium sulphate, concentration under reduced pressuregave crude product which was purified by flash chromatography (silicagel, ethyl acetate:heptane 0-50%) to give1-((3aR,4R,6R,6aS)-6-(((tert-butyldimethylsilyl)oxy)methyl)-2,2-dimethyltetrahydrothieno[3,4-d][1,3]dioxol-4-yl)-5-methylpyrimidine-2,4(1H,3H)-dione(2.9 g, 66%). ¹H NMR (300 MHz, CDCl₃) δ 8.43 (s, 1H), 7.46 (d, J=1.4 Hz,1H), 6.07 (d, J=32 Hz, 1H), 5.72 (m, 2H), 3.87 (m, 2H), 3.71 (m, 1H),1.93 (s, 3H), 1.59 (s, 3H), 1.32 (s, 3H), 0.92 (s, 9H), 0.10 (s, 3H),0.09 (s, 3H).

Synthesis of4-amino-1-((3aR,4R,6R,6aS)-6-(((tert-butyldimethylsilyl)oxy)methyl)-2,2-dimethyltetrahydrothieno[3,4-d][1,3]dioxol-4-yl)-5-methylpyrimidin-2(1H)-one

A suspension of 1,2,4-triazole (6.55 g, 94.8 mmol) in acetonitrile (140ml) was cooled in an ice batch to 0° C.; phosphorus oxychloride (2.53ml, 27.1 mmol) was added dropwise followed by triethylamine (18.9 ml,135 mmol). The mixture was stirred at 0° C. for 30 min and then asolution of1-((3aR,4R,6R,6aS)-6-(((tert-butyldimethylsilyl)oxy)methyl)-2,2-dimethyltetrahydrothieno[3,4-d][1,3]dioxol-4-yl)-5-methylpyrimidine-2,4(1H,3H)-dione(2.9 g, 6.77 mmol) in acetonitrile (25 ml) was added dropwise. Thereaction was allowed to warm to room temperature and stirred for 150 minand was then partitioned between ethyl acetate and saturated sodiumbicarbonate solution. The organic layer was washed with brine, driedover magnesium sulphate, filtered and concentrated. The residue obtainedwas dissolved in dioxane (62 ml) in an autoclave; ammonium hydroxide (62ml) was added and the vessel sealed and stirred overnight at roomtemperature. The reaction mixture was partitioned between ethyl acetateand water, the organic layer was washed with brine, dried over magnesiumsulphate and concentrated under reduced pressure. Purification by flashchromatography (silica gel:methanol:ethyl acetate 1:10) giveamino-1-((3aR,4R,6R,6aS)-6-(((tert-butyldimethylsilyl)oxy)methyl)-2,2-dimethyltetrahydrothieno[3,4-d][1,3]dioxol-4-yl)-5-methylpyrimidin-2(1H)-one(2.60 g, 90%). ¹H NMR (300 MHz, CDCl₃) δ 8.74 (brs, 2H), 7.49 (s, 1H),6.00 (d, J=2.3 Hz, 1H), 4.82 (dd, J=2.3 and 5.5 Hz, 1H), 4.74 (dd, J=3.2and 5.5 Hz, 1H), 3.91 (dd, J=5.5 and 10.5 Hz, 1H), 3.81 (dd, J=6.4 and10.5 Hz, 1H), 3.65 (m, 1H), 1.91 (s, 3H), 1.56 (s, 3H), 1.27 (s, 3H),0.89 (s, 9H), 0.07 (s, 3H), 0.06 (s, 3H).

Synthesis of4-amino-1-((3aR,4R,6R,6aS)-6-(hydroxymethyl)-2,2-dimethyltetrahydrothieno[3,4-d][1,3]dioxol-4-yl)-5-methylpyrimidin-2(1H)-one

A solution of4-amino-1-((3aR,4R,6R,6aS)-6-(((tert-butyldimethylsilyl)oxy)methyl)-2,2-dimethyltetrahydrothieno[3,4-d][1,3]dioxol-4-yl)-5-methylpyrimidin-2(1H)-one(500 mg, 1.17 mmol) in tetrahydrofuran (1.4 ml) was cooled in an icebath under argon; a solution of 1M tetrabutylammonium fluoride intetrahydrofuran (1.4 ml, 1.90 mmol) was added and the mixture stirredfor 2 hrs at room temperature. The crude product was collected byfiltration, washed with tetrahydrofuran and purified by flashchromatography (silica gel; methanol:ethyl acetate 1:10) to give4-amino-1-((3aR,4R,6R,6aS)-6-(hydroxymethyl)-2,2-dimethyltetrahydrothieno[3,4-d][1,3]dioxol-4-yl)-5-methylpyrimidin-2(1H)-one(436 mg, quant). ¹H NMR (300 MHz, DMSO) δ 7.64 (s, 1H), 7.38 (brs, 1H),6.85 (brs, 1H), 5.97 (d, J=2.8 Hz, 1H), 5.19 (t, J=5.5 Hz, 1H), 4.88(dd, J=2.8 and 5.35 Hz, 1H), 4.80 (dd, J=3.2 and 5.9 Hz, 1H), 3.67 (m,1H), 3.54 (m, 1H), 3.47 (td, J=2.8 and 6.4 Hz, 1H), 1.80 (s, 3H), 1.43(s, 3H), 1.21 (s, 3H).

Synthesis of Nucleotide Targets:

The synthesis of the nucleoside 5′-triphosphates is shown above.Nucleoside A is reacted with phosphoramidite reagent in presence ofimidazole·HCl/imidazole in dimethylformamide followed by subsequentoxidation of the phosphorous with H₂O₂ to give B in good yield (afterpurification by column chromatography on silica gel, typically 60 to 83%yield). Cleavage of the protecting groups by treatment withtrifluoroacetic acid in H₂O/dichloromethane gives the monophosphate C,typically in quantitative yield. Finally the triphosphate D is obtainedusing a method developed by Bogachev (Bogachev, V. S. Synthesis ofdeoxynucleoside 5′-triphosphates using trifluoroacetic anhydride asactivation reagent. Russ. J. Bioorg. Chem., 1996, 22, 599-604).

General Experimental Procedures:

Synthesis of B

To a solution of A (I eq.), imidazole·HCl (1.5 eq.) and imidazole (Ieq.) in dimethylformamide (3 ml/mmol of A) is added dropwisedi-tert-butyl diisopropylphosphoramidite (1.5 eq.) at room temperatureunder argon. The reaction mixture is stirred until complete consumptionof starting material was observed (LC-MS or TLC) (typically 30-90 min).The reaction mixture is then cooled in an ice-water bath and treateddropwise with 35% H₂O₂ (2.6 eq.) The reaction mixture is warmed to roomtemperature and stirred until complete reaction is observed (LC-MS orTLC); it is then cooled in an ice-water bath and carefully quenched withsaturated aqueous sodium thiosulphate. The product is extracted withethyl acetate; the organic layer is washed with brine, dried overmagnesium sulphate, filtered and concentrated under reduced pressure.Purification by column chromatography on silica gel with an appropriateeluent (generally methanol/dichloromethane) gives B, typically in 60 to85% yield.

Synthesis of C

A mixture of B (1 eq.) in dichloromethane (2 ml/mmol of B), water (3ml/mmol of B) and trifluoroacetic acid (3 ml/mmol of B) is stirredovernight at room temperature. The reaction mixture is concentratedunder reduced pressure (water bath <50° C.) to give C, typically inquantitative yield.

Synthesis of D

A cooled solution (ice-water bath) of trifluoroacetic anhydride (5 eq.)in acetonitrile (0.3 ml/mmol of trifluoroacetic anhydride) is addeddropwise to a cooled suspension of C (1 eq.) in acetonitrile (4 ml/mmolof C), triethylamine (1 eq.) and N,N-dimethylaniline (4 eq.) underargon. The reaction is then allowed to warm to room temperature, stirredat RT for 30 min and the volatiles are removed under reduced pressure.

The resulting syrup is dissolved in acetonitrile (4 ml/mmol of C),cooled in an ice-water bath and 1-methylimidazole (3 eq.) andtriethylamine (5 eq.) are added under argon. The reaction mixture isstirred for 15 min and then allowed to warm to room temperature.

A solution of tris(tetrabutylammonium)pyrophosphate (1.5 eq.) inacetonitrile (1 ml/mmol of pyrophosphate) under argon is added dropwiseat room temperature and the mixture is stirred at room temperature for45 min. The reaction is then quenched with deionised water (ca. 10-15ml/mmol of C) and stirred for 1 h. The mixture is washed with chloroform(3×10 ml), the combined organic layers are back extracted once withdeionised water (5 ml). The combined aqueous layers are directly loadedonto a column packed with DEAE Sepharose fast flow and eluted with agradient of triethylammonium bicarbonate buffer from 0.01M to 0.5M. Theproduct-containing fractions are combined and freeze dried. Theresulting nucleoside triphosphate triethylamine salt is dissolved indeionised water and then subjected to a Dowex 50 W 8 ion exchangecolumn. The fractions that show UV activity are combined and the wateris removed by freeze drying to give the nucleoside 5′-triphosphate asits sodium salt.

((2R,3S,4R,5R)-5-(2,4-Dioxo-3,4-dihydropyrimidin-1(2H)-yl)-3,4-dihydroxytetra-hydrothiophen-2-yl)methyltriphosphate sodium salt

Using the method described above for the conversion of A to B, 10 (935mg, 3.11 mmol) was converted to 14 (1.25 g, 81%). ¹H NMR (300 MHz,CDCl₃) δ 8.38 (brs, 1H), 7.74 (d, J=8.3 Hz, 1H), 6.05 (s, 1H), 5.79 (d,J=8.3 Hz), 4.85 (m, 2H), 4.19 (m, 2H), 3.83 (t, J=5.0 Hz, 1H), 1.54 (s,3H), 1.49 (s, 18H), 1.31 (s, 3H).

Using the method described above for the conversion of B to C, 14 (1.25g, 2.58 mmol) was converted to 15 (0.9 g quant). ¹H NMR (300 MHz, D₂O) δ8.13 (d, J=7.8 Hz, 1H), 5.81 (d, J=5.5 Hz, 1H), 5.76 (d, J=8.3 Hz, 1H),4.24 (dd, J=4.1 and 6.0 Hz, 1H), 4.11 (t, J=4.1 Hz, 1H), 3.98 (m, 2H),3.43 (m, 1H).

Using the method described above for the conversion of C to D, 15 (200mg, 0.59 mmol) was converted to 4′-Thio-UTP (215 mg, 62% (if 4Na+salt)). ¹H NMR (300 MHz, D₂O) δ 8.14 (d, J=8.3 Hz, 1H), 5.85 (d, J=6.4Hz, 1H), 5.81 (d, J=7.8 Hz, 1H), 4.31 (dd, J=3.7 and 6.4 Hz, 1H), 4.24(t, J=3.7 Hz, 1H), 4.12 (m, 1H), 4.00 (m, 1H), 3.44 (m, 1H). ³¹P NMR(300 MHz, D₂O) δ −8.57 (d), −10.91 (d), −22.09 (t). HPLC/MS: RT 9.638min, m/z 501 (M+H)⁺.

(((2R,3S,4R,5R)-5-(4-amino-5-methyl-2-oxopyrimidin-(2H)-yl)-3,4-dihydroxytetra-hydrothiophen-2-yl)methyltriphosphate) sodium salt

Using the method described above for the conversion of A to B, 13 (400mg, 1.28 mmol) was converted to 16 (910 mg 65%). ¹H NMR (300 MHz, CDCl₃)δ 8.19 (brs, 2H), 7.45 (s, 1H), 5.92 (d, J=2.1 Hz, 1H), 4.98 (dd, J=1.8and 6.0 Hz, 1H), 4.95 (dd, J=2.3 and 5.5 Hz, 1H), 4.24 (m, 2H), 3.80(td, J=1.8 and 6.0 Hz, 1H), 1.99 (s, 3H), 1.56 (s, 3H), 1.49 (s, 9H),1.48 (s, 9H), 1.29 (s, 3H).

Using the method described above for the conversion of B to C, 16 (480mg, 0.977 mmol) was converted to 17 (350 mg, quant). ¹H NMR (300 MHz,D₂O) δ 8.16 (s, 1H), 5.80 (d, J=6.0 Hz, 1H), 4.25 (dd, J=4.1 and 5.5 Hz,1H), 4.10 (t, J=4.6 Hz, 1H), 4.00 (m, 2H), 3.45 (m, 1H), 1.92 (s, 3H).³¹P NMR (300 MHz, D₂O) δ 0.41.

Using the method described above for the conversion of C to D, 15 (300mg, 0.849 mmol) was converted to 4′-Thio-5-MethylCTP (136 mg, 28% (if4Na+ salt)). ¹H NMR (300 MHz, D₂O) δ 7.84 (s, 1H), 5.85 (d, J=6.4 Hz,1H), 4.23 (m, 2H), 4.09 (m, 1H), 3.97 (m, 1H), 3.40 (m, 1H), 1.82 (s,3H). ³¹P NMR (300 MHz, D₂O) δ −8.96 (d), −11.00 (d), −22.28 (t). ¹³C NMR(300 MHz, D₂O) δ 165.52 (Cq), 158.09 (Cq), 139.80 (CH), 105.40 (Cq),77.26 (CH), 73.25 (CH), 66.06 (CH), 64.04 (CH2), 50.09 (CH), 12.33(CH3). HPLC/MS: RT 10.280 min, m/z 514 (M+H)⁺.

Using a procedure analogous to the preceding synthesis of4′-Thio-5-MethylCTP, but substituting 13.1 for 13, 4′-Thio-CTP can bemade.

((2R,3S,4R,5R)-5-(6-amino-9H-purin-9-yl)-3,4-dihydroxytetrahydrothiophen-2-yl)methyltriphosphate sodium salt

Using the method described above for the conversion of A to B, 19 (504mg, 1.81 mmol) was converted to 20 (444 mg, 52%). ¹H NMR (300 MHz,CDCl₃) δ 8.24 (s, 1H), 8.19 (brs, 1H), 7.71 (brs, 1H), 7.09 (s, 1H),6.20 (brs, 2H), 6.01 (d, J=5.0 Hz, 1H), 4.76 (m, 1H), 4.50 (m, 1H), 4.23(m, 2H), 3.77 (m, 1H), 1.48 (s, 18H).

Using the method described above for the conversion of B to C, 20 (430mg, 0.904 mmol) was converted to 21 (330 mg quant). ¹H NMR (300 MHz,D₂O) δ 8.64 (s, 1H), 8.26 (s, 1H), 5.86 (d, J=5.5 Hz, 1H), 4.54 (m, 1H),4.26 (m, 1H), 4.06 (m, 2H), 3.54 (m, 1H).

Using the method described above for the conversion of C to D, 21 (82mg, 0.162 mmol) was converted to 4′-Thio-ATP (35 mg, 26% (if 4Na+salt)). ¹H NMR (300 MHz, D₂O) δ 8.49 (s, 1H), 8.03 (s, 1H), 5.76 (d,J=5.6 Hz, 1H), 4.54 (dd, J=3.7 and 5.6 Hz, 1H), 4.35 (t, J=4.1 Hz, 1H),4.13 (m, 2H), 3.54 (dd, J=4.1 and 8.7 Hz, 1H). ³¹P NMR (300 MHz, D₂O) δ−9.07 (d), −10.81 (d), −22.15 (t). HPLC/MS: RT 10.467 min, m/z 524(M+H)⁺.

5-((3R,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)tetrahydrothiophen-2-yl)pyrimidine2,4(1H,3H) dione (4′-S-Pseudouridine). This compound can be madefollowing the scheme below using procedures known to those skilled inthe art.

((2R,3S,4R,5S)-5-(2,4-dioxo-1,2,3,4-tetrahydropyrimidin-5-yl)-3,4-dihydroxytetrahydrothiophen-2-yl)methyltriphosphate sodium salt (4′-Thio-ψUTP). This compound can be madefollowing the scheme below using procedures known to those skilled inthe art.

((2R,3S,4R,5R)-3,4-dihydroxy-5-(4-oxo-2-thioxo-3,4-dihydropyrimidin-1(2H)-yl)tetrahydrothiophen-2-yl)methyltetrahydrogen triphosphate (4′-S-2-S-UTP). This compound can be madefollowing the scheme below using procedures known to those skilled inthe art.

((2R,3S,4R,5R)-5-(2-amino-6-oxo-1H-purin-9(6H)-yl)-3,4-dihydroxytetrahydrothiophen-2-yl)methyltetrahydrogen triphosphate (4′-S-GTP). This compound can be madefollowing the scheme below using procedures known to those skilled inthe art.

The specification is most thoroughly understood in light of theteachings of the references cited within the specification. Theembodiments within the specification provide an illustration ofembodiments of the invention and should not be construed to limit thescope of the invention. The skilled artisan readily recognizes that manyother embodiments are encompassed by the invention. All publications andpatents cited in this disclosure are incorporated by reference in theirentirety. To the extent the material incorporated by referencecontradicts or is inconsistent with this specification, thespecification will supersede any such material. The citation of anyreferences herein is not an admission that such references are prior artto the present invention.

Unless otherwise indicated, all numbers expressing quantities ofingredients, reaction conditions, and so forth used in thespecification, including claims, are to be understood as approximationsand may vary depending upon the desired properties sought to be obtainedby the present invention. At the very least, and not as an attempt tolimit the application of the doctrine of equivalents to the scope of theclaims, each numerical parameter should be construed in light of thenumber of significant digits and ordinary rounding approaches. Therecitation of series of numbers with differing amounts of significantdigits in the specification is not to be construed as implying thatnumbers with fewer significant digits given have the same precision asnumbers with more significant digits given.

The use of the word “a” or “an” when used in conjunction with the term“comprising” in the claims and/or the specification may mean “one,” butit is also consistent with the meaning of “one or more,” “at least one,”and “one or more than one.” The use of the term “or” in the claims isused to mean “and/or” unless explicitly indicated to refer toalternatives only or the alternatives are mutually exclusive, althoughthe disclosure supports a definition that refers to only alternativesand “and/or.”

Unless otherwise indicated, the term “at least” preceding a series ofelements is to be understood to refer to every element in the series.Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, the preferred methodsand materials are now described.

The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the present application. Nothingherein is to be construed as an admission that the present invention isnot entitled to antedate such publication by virtue of prior invention.Further, the dates of publication provided may be different from theactual publication dates which may need to be independently confirmed.

Other embodiments of the invention will be apparent to those skilled inthe art from consideration of the specification and practice of theinvention disclosed herein. It is intended that the specification andexamples be considered as exemplary only, with a true scope and spiritof the invention being indicated by the following claims.

1.-18. (canceled)
 19. A lipid nanoparticle comprising: at least onefull-length mRNA molecule, wherein said full-length mRNA molecule has acoding region and, optionally, one or more non-coding regions, encodes afull length protein, and is at least 500 nucleotide residues in length,and wherein 1%-20% of the total mRNA nucleotide residues of thefull-length mRNA incorporate a 4′-thio-substituted furanose ring; andwherein said lipid nanoparticle comprises a cationic lipid, anon-cationic lipid, a cholesterol-based lipid, and a PEGylated lipid;and wherein said mRNA is encapsulated within said lipid nanoparticle.20.-22. (canceled)
 23. The lipid nanoparticle of claim 19, wherein thelipid nanoparticle comprises: a non-cationic lipid selected fromselected from DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine), DPPC(1,2-dipalmitoyl-sn-glycero-3-phosphocholine), DOPE(1,2-dioleyl-sn-glycero-3-phosphoethanolamine), DPPE(1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine), DMPE(1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine), and DOPG(,2-dioleoyl-sn-glycero-3-phospho-(1′-rac-glycerol)); and acholesterol-based lipid that is cholesterol. 24.-27. (canceled)
 28. Amethod of producing a protein in vivo, comprising administering thelipid nanoparticle of claim 19 to a subject.
 29. The method of claim 28,wherein the method produces a therapeutic protein in vivo.
 30. Apharmaceutical composition comprising the lipid nanoparticle of claim19.
 31. The lipid nanoparticle of claim 19, wherein the mRNA furthercomprises at least one nonstandard nucleotide residue.
 32. The lipidnanoparticle of claim 31, wherein the mRNA further comprises at leastone nonstandard nucleotide residue selected from the group consistingof: 5-methyl-cytidine (“5mC”), pseudouridine (“ψU”), 2-thio-uridine(“2sU”), 5-methylcytosine, isocytosine, pseudoisocytosine,5-bromouracil, 5-propynyluracil, 6-aminopurine, 2-aminopurine, inosine,diaminopurine, and 2-chloro-6-aminopurine cytosine.
 33. The lipidnanoparticle of claim 31, wherein the mRNA further comprises at leastone nonstandard nucleotide residue having a sugar modification that is a2′-O-alkyl modification.
 34. The lipid nanoparticle of claim 33, whereinthe 2′-O-alkyl modification is a 2′-deoxy-2′-fluoro modification, a2′-O-methyl modification, a 2′-O-methoxyethyl modification or a 2′-deoxymodification.